AU712901B2

AU712901B2 - Recombinant proteins of a pakistani strain of hepatitis E and their use in diagnostic methods and vaccines

Info

Publication number: AU712901B2
Application number: AU38309/95A
Authority: AU
Inventors: Suzanne U Emerson; Robert H. Purcell; Sergei A Tsarev
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1994-10-03
Filing date: 1995-10-03
Publication date: 1999-11-18
Anticipated expiration: 2015-10-03
Also published as: AU3830995A; NO326399B1; JPH11500001A; JP3889808B2; ATE518879T1; WO1996010580A2; NO971529D0; KR970706300A; EP0784631A1; CN1148450C; WO1996010580A3; EP0784631B1; NO971529L; CN1168698A; KR100359526B1; NZ295056A

Abstract

A strain of hepatitis E virus from Pakistan (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, is disclosed. The invention relates to the expression of the whole structural region of SAR-55, designated open reading frame 2 (ORF-2), in a eukaryotic expression system. The expressed protein is capable of forming HEV virus-like particles which can serve as an antigen in diagnostic immunoassays and as an immunogen or vaccine to protect against infection by hepatitis E.

Description

IA 1 Title of the Invention RECOMBINANT PROTEINS OF A PAKISTANI STRAIN OF HEPATITIS E AND THEIR USE IN DIAGNOSTIC METHODS AND VACCINES Field Of Invention The invention is in the field of hepatitis virology. More specifically, this invention relates to recombinant proteins derived from an enterically transmitted strain of hepatitis E from Pakistan, and to diagnostic methods and vaccine applications which employ these proteins.

.*15 Background of Invention Epidemics of hepatitis E, an enterically transmitted non-A/non-B hepatitis, have been reported in Asia, Africa and Central America (Balayan, M.S. (1987), Soviet Medical Reviews, Section E, Virology Reviews, Zhdanov, 0- V.M. Chur, Switzerland: Harwood Academic Publishers, vol. 2, 235-261; Purcell, et al. (1988) in Zuckerman, A.J. "Viral Hepatitis and Liver Disease", New York: Alan R. Liss, 131-137; Bradley, D.W.

(1990), British Medical Bulletin, 46:442-461; Ticehurst, J.R. (1991) in Hollinger, Lemon, Margolis, H.S. (eds): "Viral Hepatitis and Liver Disease", Williams and Wilkins, Baltimore, 501-513). Cases of sporadic hepatitis, presumed to be hepatitis E, account for up to of reported hepatitis in countries where hepatitis E virus (HEV) is endemic. The need for development of a serological test for the detection of anti-HEV antibodies in the sera of infected individuals is widely recognized in the field, but the very low concentration of HEV excreted from infected humans or animals made it impossible to use such HEV as the source of antigen for

V';

1 WO 96/10580 PCT/US95/13102 2 0 serological tests and although limited success was reported in propagation of HEV in cell culture (Huang, R.T. et al. (1992), J. Gen. Virol., 73:1143-1148), cell culture is currently too inefficient to produce the amounts of antigen required for serological tests.

Recently, major efforts worldwide to identify viral genomic sequences associated with hepatitis E have resulted in the cloning of the genomes of a limited number of strains of HEV (Tam, A.W. et al. (1991), Virology, 185:120-131; Tsarev, S.A. et al. (1992), Proc. Natl. Acad.

Sci. USA, 89:559-563; Fry, K.E. et al. (1992), Virus Genes, 6:173-185). Analysis of the DNA sequences have led investigators to hypothesize that the HEV genome is organized into three open reading frames (ORFs) and to 1 hypothesize that these ORFs encode intact HEV proteins.

A partial DNA sequence of the genome of an HEV strain from Burma (Myanmar) is disclosed in Reyes et al., 1990, Science, 247:1335-1339. Tam et al., 1991, and Reyes et al., PCT Patent Application W091/15603 published S October 17, 1991 disclose the complete nucleotide sequence 2 and a deduced amino acid sequence of the Burma strain of HEV. These authors hypothesized that three forward open reading frames (ORFs) are contained within the sequence of this strain.

Ichikawa et al., 1991, Microbiol. Immunol., 2 35:535-543, discloses the isolation of a series of clones of 240-320 nucleotides in length upon the screening of a Xgtll expression library with sera from HEV-infected cynomolgus monkeys. The recombinant protein expressed by one clone was expressed in E. coli. This fusion protein 3 is encoded by the 3' region of ORF-2 of the Myanmar strain of HEV.

The expression of additional proteins encoded within the 3' region of ORF-2 of a Mexican strain of HEV and of a Burmese strain of HEV is described in Yarbough et al., 1991 J. Virology, 65:5790-5797. This article WO 96/10580 PCTUS95/13102 3 0 describes the isolation of two cDNA clones derived from HEV. These clones encode the proteins in the 3' region of ORF-2. The clones were expressed in E. coli as fusion proteins.

Purdy et al., 1992, Archives of Virology, 123:335-349, and Favorov et al., 1992, J. of Medical Virology, 36:246-250, disclose the expression of a larger ORF-2 protein fragment from the Burma strain in E. coli.

These references, as well as those previously discussed, only disclose the expression of a portion of the ORF-2 gene using bacterial expression systems. Successful expression of the full-length ORF-2 protein has not been disclosed until the present invention.

Comparison of the genome organization and S morphological structure of HEV to that of other viruses 1 revealed that HEV is most closely related to the caliciviruses. Of interest, the structural proteins of caliciviruses are encoded by the 3' portion of their genome (Neil, J.D. et al. (1991) J. Virol., 65:5440-5447; and Carter, M.J. et al. (1992), J. Arch. Virol., 122:223- 2 235) and although there is no direct evidence that the 3' terminal part of the HEV genome also encodes the structural proteins, expression of certain small portions of the 3' genome region in bacterial cells resulted in production of proteins reactive with anti-HEV sera in 2 ELISA and Western blots (Yarborough, et al., (1991); Ichikawa et al. (1991); Favorov et al. (1992) and Dawson, G.J. et al. (1992) J. Virol. Meth; 38:175-186). However, the function of ORF-2 protein as a structural protein was not proven until the present invention.

The small proteins encoded by a portion of the ORF-2 gene have been used in immunoassay to detect antibodies to HEV in animal sera. The use of small bacterially expressed proteins as antigens in serological immunoassays has several potential drawbacks. First, the expression of these small proteins in bacterial cells often results in solubility problems and in non-specific cross-reactivity of patients' sera with E. coli proteins when crude E. coli lysates are used as antigens in immunoassays (Purdy et al. (1992)). Second, the use of Western blots as a first-line serological test for anti- HEV antibodies in routine epidemiology is impractical due to time and cost constraints. An ELISA using smallpeptides derived from the 3'-terminal part of the HEV genome resulted in the detection of only 41% positives 0.:1 from known HEV-infected patients. Third, it has been shown that for many viruses, including Picornaviridae which is the closest family to the Caliciviridae, important antigenic and immunogenic epitopes are highly conformational (Lemon, S.M. et al. (1991), in Hollinger, Lemon, Margolis, H.S. (eds): "Viral Hepatitis and Liver Disease", Williams and Wilkins, Baltimore, 24). For this reason, it is believed that expression in a eukaryotic system of a complete ORF encoding an intact HEV gene would result in production of a protein which could form HEV-virus-like particles. Such a complete ORF 20 S protein would have an immunological structure closer to that of native capsid protein(s) than would the abovenoted smaller proteins which represent only portions of the structural proteins of HEV. Therefore, these complete ORF proteins would likely serve as a more representative 25 5 antigen and a more efficient immunogen than the currentlyused smaller proteins.

Summary Of Invention The present invention relates to an isolated and substantially pure preparation of a human hepatitis E viral strain i An aspect of the present invention is a nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of a hepatitis E virus open reading frame 2 protein. A nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of SEQ ID No. 2.

In addition the present invention provides recombinant vectors comprising the said nucleic acid molecule and host cells transferred, transfested or infected in vitro with such vectors.

An aspect of the present invention is a method for producing a hepatitis E virus open reading frame 2 protein comprising: culturing a host cell containing a nucleic acid molecule g. consisting of nucleotides which encode amino acids 112-660 of a hepatitis E Svirus open reading frame 2 protein under conditions appropriate to cause expression of said protein; and

S

obtaining said expressed protein from the host cell.

Another method of the present invention a method for producing a hepatitis E virus open reading frame 2 protein comprising: culturing a host cell containing a nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of SEQ ID No:2 under conditions appropriate to cause expression of said protein; and 2 obtaining said expressed protein from the host cell.

U A further aspect of the present invention is a purified and isolated hepatitis E virus open reading frame 2 protein produced by the method S° according to claim 6, wherein said protein has a molecular weight of approximately 55 kilodaltons and its amino terminus at amino acid 112 of a hepatitis E virus open reading frame 2 protein. A purified and isolated hepatitis E virus open reading frame 2 protein produced by the method of claim 17, wherein said protein has a molecular weight of approximately 55 kilodaltons and its amino terminus at amino acid 112 of SEQ ID No: 2.

The present invention provide a method of detecting antibodies to hepatitis E virus in a biological sample, said method comprising: VV :nwo rdVioletPhil\Nodelete%38309-95 doc A k 6 contacting said sample with the hepatitis E virus protein according to any one of claims 8 to 11 to form an immune complex with the antibodies; and detecting the presence of the immune complex.

The present invention provides a vaccine for immunizing a mammal against hepatitis E, wherein said vaccines comprises the protein as hereinbefore described.

Furthermore, the protein of the present invention can be used for the manufacture use of a protein according to any one of claims 8 to 11 for the manufacture of a medicament for use in a method of preventing hepatitis E in a mammal, comprising administering the medicament to the mammal in an amount effective to stimulate the production of protective antibodies.

The present invention also encompasses methods of detecting antibodies specific for hepatitis E virus in biological samples. Such methods 5 are useful for diagnosis of infection and disease caused by HEV, and for monitoring the progression of such disease. Such methods are also useful for monitoring the efficacy of therapeutic agents during the course of treatment of e 0 HEV infection and disease in a mammal.

*o0 The present invention provides a method of detecting hepatitis E virus in 20 a biological sample, said method comprising: S. contacting said sample with the antibodies according to any one of claims 17 to 19 to form an immune complex with the hepatitis E virus; and 9 detecting the presence of the immune complex.

Other aspects of the present invention include a kit and a pharmaceutical composition comprising the proteins as hereinbefore described.

Therefore this invention also relates to pharmaceutical compositions for use in prevention or treatment of Hepatitis E in a mammal.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is '";pnot intended to exclude other additives, components, integers or steps.

6 r VA :WinwordViolet\PhlrNodelete38309-95 doc Description Of Figures Figure 1 shows the recombinant vector used to express the complete ORF-2 protein of the genome of HEV strain Figures 2A and 2B are sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE) in which cell lysates of insect cells infected with wild-type baculovirus or recombinant baculovirus (containing the gene encoding

ORF-

2) were either stained with Coomassie blue or subjected to Western blotting with serum of an HEVinfected chimp In both Figures 2A and 2B, lane 1 contains total cell lysate of noninfected SF-9 cells; lane 2 contains lysate of cells infected with wild-type o: baculovirus; lane 3 contains lysate of cells infected with recombinant baculovirus and lane 4 contains molecular weight markers.

.6 Figures 3A-3A' and 3B show immunoelectron micrographs (IEM) of 30 and 20 nm virus-like particles respectively, which are formed as a result of the expression of ORF-2 protein in recombinant infected insect cells.

Figure 4 shows the results of an ELISA using as the antigen, recombinant ORF-2 which was expressed from insect cells containing the gene encoding the complete ORF-2. Serum anti-HEV antibody levels were determined at various times following inoculation of cynomolgus monkeys with either the Mexican (Cyno-80A82, Cyno-9A97 and Cyno 83) or Pakistani (Cyno-374) strains of HEV.

Figures 5A-D show the results of an ELISA using WO 96/10580 PCT/US95/13102 7 as the antigen, recombinant ORF-2 which was expressed from insect cells containing the gene encoding the complete ORF-2. Serum IgG or IgM anti-HEV levels were determined over time following inoculation of two chimpanzees with

HEV.

Figures 6A-J show a comparison of ELISA data obtained using as the antigen the recombinant complete ORF-2 protein derived from SAR-55 as the antigen vs. a recombinant partial ORF-2 protein derived from the Burma strain of HEV (Genelabs).

Figures 7A-J show anti-HEV IgG ELISA and alanine aminotransferase (ALT) values for cynomolgus monkeys inoculated with ten-fold serial dilutions (indicated in parenthesis at the top of each panel),of a 10% fecal S suspension of SAR-55 HEV. Recombinant antigens used in ELISA were: glutathione-S-transferase (GST); a fusion of the 3-2 epitope [Yarbough et al., (1991)

J.

Virol, 65:5790-5797] and GST; SG3 a fusion of 327 Cterminal amino acids of ORF-2 and GST [Yarbough et al., (1993): Assay Development of diagnostic tests for 2 Hepatitis E in "International Symposium on Viral Hepatitis and Liver Disease. Scientific Program and Abstract Volume." Tokyo:VHFL p. 87]; and a 55 kDa ORF-2 product directly expressed in insect cells.

Figures 8A-E show anti-HEV IgM ELISA and ALT 2 values for positive cynomolgus monkeys inoculated with ten-fold serial dilutions (indicated in parenthesis at the top of each panel) of the 10% fecal suspension of HEV. Recombinant antigens used in ELISA were: S glutathione-S-transferase (GST); a fusion of the 3 3-2 epitope [Yarbough et al., 1991] and (GST); SG3 a fusion of 327 C-terminal amino acids of ORF-2 and GST [Yarbough et al., 1993]; and the 55 kDa ORF-2 product directly expressed in insect cells.

Figure 9 shows an ethidium bromide stain of a 2% agarose gel on which PCR products produced from extracts WO 96/10580 PCT/US95/13102 8 0 of serial ten-fold dilutions (indicated at the top of each lane of the gel) of the 10% fecal suspension of the HEV were separated. The predicted length of the PCR products was about 640 base pairs and the column marked with an contains DNA size markers.

Figure 10 shows the pPIC9 vector used to express the complete ORF-2 protein or lower molecular weight fragments in yeast.

Detailed Description of Invention The present invention relates to an isolated and substantially purified strain of hepatitis E virus (HEV) from Pakistan, SAR-55. The present invention also relates to the cloning of the viral genes encoding proteins of HEV and the expression of the recombinant proteins using an expression system. More specifically, the present invention relates to the cloning and expression of the open reading frames (ORF) of HEV derived from The present invention relates to isolated HEV proteins. Preferably, the HEV proteins of the present invention are substantially homologous to, and most 20 preferably biologically equivalent to, the native

HEV

proteins. By "biologically equivalent" as used throughout the specification and claims, it is meant that the compositions are capable of forming viral-like particles and are immunogenic. The HEV proteins of the present invention may also stimulate the production of protective antibodies upon injection into a mammal that would serve to protect the mammal upon challenge with a wild-type

HEV.

By "substantially homologous" as used throughout the ensuing specification and claims, is meant a degree of homology in the amino acid sequence to the native HEV proteins. Preferably the degree of homology is in excess of preferably in excess of 90%, with a particularly preferred group of proteins being in excess of 99% homologous with the native HEV proteins.

Preferred HEV proteins are those proteins that WO 96/10580 PCTIUS95/13102 9 are encoded by the ORF genes. Of particular interest are proteins encoded by the ORF-2 gene of HEV and most particularly proteins encoded by the ORF-2 gene of the strain of HEV. The preferred proteins of the present invention, encoded by the ORF-2 gene, form viruslike particles. The amino acid sequences of the ORF-1, ORF-2 and ORF-3 proteins are shown below as SEQ ID NO.: 1, SEQ ID NO.: 2, and SEQ ID NO.: 3, respectively: (SEQ. ID NO.: 1) Met Glu Ala His Gln Phe Ile Lys Ala Pro Gly Ile Thr Thr Ala 0 1 5 10 Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn 25 Ala Val Val Val Arg Pro Phe Leu Ser His Gln Gln Ile Glu Ile 40 Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu 55 5 Val Phe Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu 70 1 1.

Glu Leu Tyr Cys Ala His Pro Arg Cys Phe Leu Arg Ala Pro Thr Arg Arg Gly Leu Pro Ser Gly Cys Asn Leu His Asp Met His Gly Met Thr Val Leu Leu Pro Ile His Asp Gly Ser Ala Gly Tyr Arg Thr Thr Lys Val Arg Ala Ile Pro Glu Pro Ser Arg Ala Arg Ser Gly Arg 85 Ser Ile Asn Asp Asn Pro 100 Pro Ala Gly Arg Asp Val 110 115 Cys Leu Glu Asn Val Val Gln Arg Trp Ile His Tyr Gly Arg 105 Thr 120 Gly Pro Ala Ala Asn Cys Arg 125 130 Ala Ala Asp Arg Thr Tyr Cys 140 145 Phe Pro Ala Glu Thr Gly Ile 155 160 Ser Pro Ser Asp Val Ala Glu 170 175 Arg Leu Tyr Ala Ala Leu His 185 190 Pro Gly Thr Tyr Arg Thr Ala 200 205 Arg Arg Val Val Val Thr Tyr 215 220 Asn His Asp Val Ser Asn Leu 230 235 Val Thr Gly Asp His Pro Leu 245 250 Gly Cys His Phe Val Leu Leu 260 265 Pro Met Pro Tyr Val Pro Tyr 275 280 Arg Ser Ala Leu 135 Phe Asp Gly Phe 150 Ala Leu Tyr Ser 165 Ala Met Phe Arg 180 Leu Pro Pro Glu 195 Ser Tyr Leu Leu 210 Glu Gly Asp Thr 225 Arg Ser Trp Ile 240 Val Ile Glu Arg 255 Leu Thr Ala Ala 270 Pro Arg Ser Thr 285 Gly Thr Pro Ser 300 Glu Val Tyr Val Arg Ser Ile Phe Gly Pro Gly 290 295 WO 96/10580 PCTIUS9SI13 102 0 10 Leu Phe Pro Thr Ser Cys Ser Thr Lys Pro Ala His Asp Asp Gin Gly Ile Ser Gly Trp Asn Ala Tyr Leu Ile Ser Lys Phe Ile Thr Asp Tyr Ile Arg Trp Leu Phe Asp Giu Ile Al a Tyr Al a Trp Asp Ar 320 Phe Cys Cy 335 Lys Val Tb: 350 Ser Giu Asj 5 r 365 Tb: Gl~ Arc Prc Sey Ser Ala Val Ser Lys Cys Thr Cys Phe Gly His Asp Asn Glu Ser Ala Ile Thr Ala Leu Gin Ile Val Ala Arg Gin Val Asp Gly Thr Phe Arg Thr Gly Pro Glu Arg Met Ala Ala Gly rle Cys Hi 380 SMet Arg Ar 395 Leu Tyr Se 410 Gly Arg Gil 425 Ala Giy Ph 440 Ala Pro CyE 455 Phe Cys CyE 470 Leu Gin Prc 485 Glu Ala TIyx 500 Ser Asp Ile 515 Pro Leu Tyr 530 Ala Gly Arg 545 Arg Ile Asp 560 Ser Phe Val 575 His Asn Leu 590 Pro Phe Ser 605 Arg Tyr Val 620 Gly Val Ser 635 Ser Ala Leu 5 r n Leu Ser Val Ala Gin Leu Trp Leu His His Phe Ala Glu Ser Glm I Leu Met Arg Gly Leu Arg Giu Leu Giu Leu Cys Met G1u fly lia 2hr Ser Thr Phe His Ala Val 310 315 Leu Phe Gly Ala Tbr Leu 325 330 Leu Met Thr Tyr Leu Arg 340 345 Thr Leu Val Ala Asn Glu 355 360 Thr Ala Val Ile Thr Ala 370 375 Tyr Leu Arg Thr Gin Ala 385 390 Arg Giu His Ala Gin Lys 400 405 Phe Giu Lys Ser Gly Arg 415 420 Phe Tyr Ala Gin Cys Arg 430 435 Asp Pro Arg Val Leu Val 445 450 Arg Thr Ala Ile Arg Lys 460 465 Lys Trp Leu Gly Gin Glu 475 480 Gly Val Val Gly Asp Gin 490 495 Ser Asp Val Asp Pro Ala 505 510 Ser Tyr Val Val Pro Gly 520 525 Ueu Asp Leu Pro Ala Glu 535 540 kla Thr Val Lys Val Ser Gly Leu Val Phe Thr Ala Giu Ala Phe Val Pro Cys DDU 555 Cys Glu Thr Leu Leu Gly Asn Lys 565 570 Asp Giy Ala Val Leu Giu Thr Asn 580 585 Ser Phe Asp Ala Ser Gln Ser Thr 595 600 Leu Thr Tyr Ala Ala Ser Ala Ala 610 615 Ala Ala Gly Leu Asp His Arg Ala 625 630 Pro Arg Ser Ala Pro Gly Glu Val 640 645 Tyr Arg Phe Asn Arg Giu Ala Gin 655 .660 Phe Trp Phe His Pro Giu Gly Leu 670 675 Ser Pro Gly His Val Trp Giu Ser 685 690 Ser Thr Leu Tyr Thr Arg Thr Trp 700 705 650 Arg Leu Leu Gly Ala Asn Ser Pro Pro Leu Thr Gly 665 Phe Ala Pro 680 Phe Cys Gly 695 Asn Phe Glu WO 96/10580 PCr[US9S/13102 11 Ser Glu Val Asp Ala Val Pro Ser Pro 710 Phe Thr Ser Glu Pro Ser Ile Pro 725 Pro Ala Ala Pro Leu Pro Pro Pro 740 Leu Ser Ala Pro Ala Arg Gly Glu 755 Arg Ala Pro Ala Ile Thr His Gin 770 Leu Phe Thr Tyr Pro Asp Gly Ser 785 Phe Glu Ser Thr Cys Thr Trp Leu 800 His Arg Pro Gly Gly Gly Leu Cys 815 Pro Ala Ser Phe Asp Ala Ala Ser 830 Ala Ala Tyr Thr Leu Thr Pro Arg 845 Pro Asp Tyr Arg Leu Glu His Asn 860 Tyr Arg Glu Thr Cys Ser Arg Leu 875 Leu Gly Thr Gly Ile Tyr Gin Val 890 Ala Trp Glu Arg Asn His Arg Pro 905 Glu Leu Ala Ala Arg Trp Phe Glu 920 Thr Leu Thr Ile Thr Glu Asp Val 935 Ile Glu Leu Asp Ser Ala Thr Asp 950 Cys Arg Val Thr Pro Gly Val Val 965 Val Pro Gly Ser Gly Lys Ser Arg E 980 Asp Val Val Val Val Pro Thr Arg C 995 Arg Arg Gly Phe Ala Ala Phe Thr I 1010 Thr Gin Gly Arg Arg Val Val Ile A 1025 Pro His Leu Leu Leu Leu His Met G 1040 Leu Leu Gly Asp Pro Asn Gin Ile P 1055 Ala Gly Leu Val Pro Ala Ile Arg P 1070 Ala Gin Pro Asp Leu Gly 715 720 Arg Ala Ala Thr Pro Thr Sei Ala Prc Thr Lys Val His Phe Pro Pro Gly Pro Gly Ala r 730 SPro Asp 745 Ala Pro 760 Ala Arg 775 SVal Phe 790 Asn Ala 805 Ala Phe 820 Val Met 835 Ile Ile 850 Lys Arg 865 Thr Ala 880 Ile Gly 895 Asp Glu 910 Asn Arg SPro Ser Pro Gly Ala Thr His Arg Arg Ala Gly Ser Ser Asn Val Tyr Gin Arg Arg Asp Gly His Ala Val Leu Glu Ala Ala Tyr Pro Pro Ser Phe Leu Tyr Leu Pro Thr Cys 735 Thr 750 Ala 765 Leu 780 Leu 795 Asp 810 Tyr 825 Ala 840 Ala 855 Ala 870 Leu 885 Asp 900 Pro 915 Pro 925 930 Ala Arg Thr Ala Asn Leu Ala 940 945 lal Gly Arg Ala Cys Ala Gly 955 960 G1n Tyr Gin Phe Thr Ala Gly 970 975 3er Ile Thr Gin Ala Asp Val 985 990 Glu Leu Arg Asn Ala Trp Arg 1000 1005 ?ro His Thr Ala Ala Arg Val 1015 1020 Isp Glu Ala Pro Ser Leu Pro 1030 1035 ~n Arg Ala Ala Thr Val His 1045 1050 'ro Ala Ile Asp Phe Glu His 1060 1065 ro Asp Leu Ala Pro Thr Ser 1075 1080 ro Ala Asp Val Cys Glu Leu 1090 1095 In Thr Thr Ser Arg Val Leu 1105 1110 Trp Trp His Val Ile Arg Gly Ala Thr His Arg Cys P 1085 Tyr Pro Met Ile G 1100 WO 96/10580 PCTIUS95/13102 12 Arg Ser Leu Phe Trp Gly Glu 1115 Phe Thr Gin Ala Ala Lys Ala 1130 His Glu Ala Gin Gly Ala Thr 1145 Thr Ala Asp Ala Arg Gly Leu 1160 Ile Val Ala Leu Thr Arg His 1175 Ala Pro Gly Leu Leu Arg Glu 1190 Asn Asn Phe Phe Leu Ala Gly 1205 Ser Val Ile Pro Arg Gly Asn 1220 Ala Ala Phe Pro Pro Ser Cys 1235 Ala Glu Glu Leu Gly His Arg 1250 Pro Pro Cys Pro Glu Leu Glu 1265 Glu Leu Thr Thr Cys Asp Ser 1280 Ile Val His Cys Arg Met Ala 1295 Leu Ser Thr Leu Val Gly Arg 1310 Asn Ala Ser His Ser Asp Val 1325 Pro Ala Ile Gly Pro Val Gn 1340 Leu Glu Glu Ala Met Val Glu 1355 Leu Glu Leu Asp Leu Cys Ser 1370 Phe Gin Lys Asp Cys Asn Lys 1385 His Gly Lys Val Gly Gin Gly 1400 Cys Ala Leu Phe Gly Pro Trp I 1415 Leu Ala Leu Leu Pro Gin Gly 1430 Asp Thr Val Phe Ser Ala Ala I Pro Ala Val Gly Gin Lys Leu Val Ala Tyr Ile Thr Val Gly Pro Glu Pro Gin Val Ala Tyr Arg Val Lys Arg 1 Phe Ile ?he Val I al A lu P ila I 1120 Asn Pro Gly Se: 1135 Thr Glu Thr Th: 1150 Gin Ser Ser Ar< 1165 Glu Lys Cys Va: 1180 Gly Ile Ser Asi 1195 Glu Ile Gly HiE 1210 Asp Ala Asn Va] 1225 Ile Ser Ala Phe 1240 Ala Pro Val Ala 1255 Gly Leu Leu Tyr 1270 Val Thr Phe Glu 1285 Pro Ser Gin Arg 1300 Gly Arg Arg Thr 1315 Asp Ser Leu Ala 1330 rhr Thr Cys Glu 1345 Gly Gin Asp Gly 1360 Asp Val Ser Arg 1375 rhr Thr Gly Glu 1390 Ser Ala Trp Ser 1405 Arg Ala Ile Glu 1420 'he Tyr Gly Asp 1435 la Ala Ala Lys 1450 'he Asp Ser Thr 1465 le Met Glu Glu 1480 1125 r Val Thr Val 1140 r Ile Ile Ala 1155 g Ala His Ala 1170 L Ile Ile Asp 1185 p Ala Ile Val 1200 SGin Arg Pro 1215 Asp Thr Leu 1230 His Glu Leu 1245 i Ala Val Leu 1260 Leu Pro Gin 1275 Leu Thr Asp 1290 Lys Ala Val 1305 Lys Leu Tyr 1320 Arg Phe Ile 1335 Leu Tyr Glu 1350 Ser Ala Val 1365 Ile Thr Phe 1380 Thr Ile Ala 1395 Lys Thr Phe 1410 Lys Ala Ile 1425 Ala Phe Asp 1440 Ala Ser Met 1455 Gin Asn Asn 1470 Cys Gly Met 1485 Ser Ala Trp 1500 Trp Lys Lys 1515 Val Phe Pro Ile 1445 Phe Glu Asn Asp Phe 1460 Ser Leu Gly Leu Glu 1475 Gin Trp Leu Ile Arg 1490 Leu Gin Ala Pro Lys 1505 Ser G Cys A Leu Tyr His Leu Ile Arg 1495 Glu Ser Leu Arg Gly Phe 1510 WO 96/10580 PCT/US9/13102 13 His Ser Gly Glu Met Ala Val Ala Ala Phe Arg Gin Ser Lys Leu Lys Val Val Ala Ala Gly Arg Ala Glu Gin Thr Asn Val Gly Val Ser Ala Val Ala Val Leu Asp Ile Lys Pro Val Pro Leu Leu Ala Pro Asp Leu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn 1520 1525 1530 Thr His Cys Tyr Asp Phe Arg Asp Leu Gin Val 1535 1540 1545 Gly Asp Asp Ser Ile Val Leu Cys Ser Glu Tyr 1550 1555 1560 Gly Ala Ala Val Leu Ile Ala Gly Cys Gly Leu 1565 1570 1575 Asp Phe Arg Pro Ile Gly Leu Tyr Ala Gly Val 1580 1585 1590 Gly Leu Gly Ala Leu Pro Asp Val Val Arg Phe 1595 1600 1605 Thr Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg 1610 1615 1620 Arg Leu Ala Val Ser Asp Phe Leu Arg Lys Leu 1625 1630 1635 Gln Met Cys Val Asp Val Val Ser Arg Val Tyr 1640 1645 1650 Gly Leu Val His Asn Leu Ile Glu Met Leu Gin 1655 1660 1665 Gly Lys Ala His Phe Thr Glu Ser Val Lys Pro 1670 1675 1680 Thr Asn Ser Ile Leu Cvs Ara Val Glu 1685 1690 (SEQ. ID NO.: 2) Met Arg Pro Arg Pro Ile Leu Leu Leu Leu Leu Met Phe Leu Pro 1 5 10 Met Leu Pro Ala Pro Pro Pro Gly Gin Pro Ser Gly Arg Arg Arg 25 Gly Arg Arg Ser Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg 40 Val Asp Ser Gin Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn 55 Pro Phe Ala Pro Asp Val Thr Ala Ala Ala Gly Ala Gly Pro Arg 65 70 Val Arg Gin Pro Ala Arg Pro Leu Gly Ser Ala Trp Arg Asp Gin 85 Ala Gin Arg Pro Ala Ala Ala Ser Arg Arg Arg Pro Thr Thr Ala 100 105 Gly Ala Ala Pro Leu Thr Ala Val Ala Pro Ala His Asp Thr Pro 110 115 120 Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gin 125 130 135 Tyr Asn Leu Ser Thr Ser Pro Leu Thr Ser Ser Val Ala Thr Gly 140 145 150 Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu Ser Pro Leu Leu Pro 155 160 165 Leu Gin Asp Gly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser 170 175 180 Asn Tyr Ala Gin Tyr Arg Val Ala Arg Ala Thr Ile Arg Tyr Arg 185 190 195 WO 96/10580 PCT/US95/13102 -14 Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser 200 205 210 Phe Tyr Pro Gin Thr Thr Thr Thr Pro Thr Ser Val Asp Met Asn 215 220 225 Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gin Pro Gly Ile 230 235 240 Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn 245 250 255 Gin Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu 260 265 270 Ala Thr Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val 275 280 285 Asn Ser Tyr Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu 290 295 300 Asp Phe Ala Leu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn 305 310 315 Thr Asn Thr Arg Val Ser Arg Tyr Ser Ser Thr Ala Arg His Arg 320 325 330 Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala 335 340 345 Ala Thr Arg Phe Met Lys Asp Leu Tyr Phe Thr Ser Thr Asn Gly 350 355 360 Val Gly Glu Ile Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu 365 370 375 Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser 380 385 390 Ala Gly Gly Gin Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn 395 400 405 Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gin 410 415 420 Gin Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp Leu Gly Glu 425 430 435 Ser Arg Val Val Ile Gin Asp Tyr Asp Asn Gin His Glu Gin Asp 440 445 450 Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu 455 460 465 Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr 470 475 480 Asp Gin Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser 485 490 495 Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gin Ala Val 500 505 510 Ala Arg Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro 515 520 525 Leu Ser Thr Ile Gin Gin Tyr Ser Lys Thr Phe Phe Val Leu Pro 530 535 540 Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala 545 550 555 Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gin Leu Leu 560 565 570 Val Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr 575 580 585 Thr Ser Leu Gly Ala Gly Pro Val Ser Ile Ser Ala Val Ala Val 590 595 600 WO 96/10580 PCTIUS95/13102 15 Leu Ala Pro His Ser Val Leu Ala Leu Leu Glu Asp Thr Met Asp 605 610 615 Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys 620 625 630 Arg Pro Leu Gly Leu Gin Gly Cys Ala Phe Gin Ser Thr Val Ala 635 640 645 Glu Leu Gin Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Leu 650 655 660 (SEQ. ID NO.: 3) Met 1 Ala Cys Gly Ile Asn Asn Met Ser 5 Leu Gly Leu Phe Pro Arg His Arg Ala Ala Ala Val Leu Ser Pro Ser Phe Ala Ala Pro Met Gly Ser 10 Cys Cys Cys Ser Ser Cys Phe 25 Pro Val Ser Arg Leu Ala Ala 40 Pro Ala Val Val Ser Gly Val 55 Gin Ser Pro Ile Phe Ile Gin 70 Pro Leu Arg Pro Gly Leu Asp 85 His Ser Ala Pro Leu Gly Val 100 Pro His Val Val Asp Leu Pro 115 Arg Pro Cys Cys Leu Cys Val Val Gly Thr Gly Leu Pro Thr Pro Leu Val Phe Thr Arg Pro 105 Gin Leu Gly 120 Ser Pro Pro Met Ser Ala Asn Pro Pro Asp Ser Ala Pro Pro Leu 110 Pro Arg Arg The three-letter abbreviations follow the conventional amino acid shorthand for the twenty naturally occurring amino acids.

The preferred recombinant HEV proteins consist of at least one ORF protein. Other recombinant proteins made up of more than one of the same or different

ORF

proteins may be made to alter the biological properties of the protein. It is contemplated that additions, substitutions or deletions of discrete amino acids or of discrete sequences of amino acids may enhance the biological activity of the HEV proteins.

The present invention is also a nucleic acid sequence which is capable of directing the production of the above-discussed HEV protein or proteins substantially homologous to the HEV proteins. This nucleic acid sequence, designated SAR-55, is set forth below as SEQ ID WO 96/10580 PCTIUS95/13102 16 NO.: 4 and was deposited with the American Type Culture Collection (ATCC) on September 17, 1992 (ATCC accession number 75302).

AGGCAGACCA

AGTTTATCAA

GGCTGCTCTA

GTGGTAGTTA

TCCTTATTAA

CCCCGAGGTT

CATAATGAGC

GCTGCCTCGA

CAATCCTAAT

GGGCGTGATG

GGCCGGCTGC

CCCCGCTGCT

GGCTGTAACT

CTCTCCATGA

GTTCCGCCAT

CTCCCGCCTG

CCGCGTCGTA

GGTGACGTAT

GATGTTTCCA.

TTACCGGAGA

CATTGGCTGC

GAGCCATCAC

CCGAGGTCTA

CCCCTCCCTA

TTCCATGCTG

TGTTCGGGGC

CCGCCTAATG

I

ACTGTGGGCA

C

CTGAGGACGC

'I

TACCATCTGCC

TCTAAGGGGA p nm i CATATGTGGT CGATGCCATG GGCTCCTGGC

ATCACTACTG

GCAGCGGCCA

ACTCTGCCCT

GGCCTTTTCT

CTCTCACCAG

CCTAATGCAA,

CCTCGCCAGC

TTCTGGAACC

ATCCCATCCA

TGGAGCTTTA

CTGTCGCGC

AATTGGTGCC

GTGGTCCACC

TTCAGCGTTG

TAATTGCCGG

GACCGCACTT

TTCCCGCCGA

TATGTCACCA,

GGTATGACGC

AGGTCCTGTT

CTTGCTGATC

GAGGGTGACA

ACCTGCGCTC

CCACCCTCTC

CACTTTGTCC

CTATGCCCTA

EGTCCGATCG

ETTCCAACCT

rCCCTGCCCA

MACCCTAGAT

~CTTACCTCC

~CCTTGTTGC

'CTTACAGCT

!ACCAGCGGT

'GCGTCGCCT

CACCCCCGCT

GTTGCTTCCT

GTATACTGCC

CGTTCCGCGC

ACTGCTTCGA

GACGGGCATC

TCTGATGTCG

GGCTTTACGC

GCCCCCTGGC

CATGACGGCA

CTAGTGCTGG

CTGGATTAGA

GTCATCGAGC

TTTTACTCAC

TGTCCCTTAC

ATCTTCGGCC

CATGCTCCAC

TATCTGGGAC

GACCAAGCCT

GCGGCATTAG

CAATGAAGGC

=TATCACTGC

kCCTCCGCAC 9]

;GAGCGGGAGC

GAGGCCCATC

CTATTGAGCA

TGCGAATGCT

CAGATTGAGA

TTGTTTTCCG

GCGTGTTATC

CGCTCCGGCC

CAATAAATGA

CCGTCCTGCC

CCTACCCGCG

TGCGCGGGCT

CGGGTTTTCT

GCCCTCTATT

CCGAGGCTAT

TGCCCTCCAC

ACATACCGCA

GGCGCGTTGT

TTATAACCAC

ACCACTAAGG

GGGTTAGGGC

GGCTGCTCCG

CCCCGGTCTA

CGGGTGGCAC

CAAGTCGACC

7GTCTCATGT

E'TTGCTGCTC

TACAAGGTT

['GGAACGCCT

XCGCCTACCT

'CAGGCTATA

:ATGCTCAGA

120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880 920 960 1000 1040 1080 1120 1160 1200 1240 1280 Mj;LWTCTAC AGTTGGCTCT TTGAGAAGTC WO 96/10580 PC/US9S/13102 17 CGGCCGTGAT TATATCCCCG GCCGTCAGTT

GGAGTTCTAC

GCTCAGTGTA

GGCGCTGGC

ACCCACGGGT

GTTGGTTTT'

CTGTAGGACT

GCGATTCGT.

TGCTTTATGA AGTGGCTGG( TACAACCTGC

AGAAGGCGT(

CAACGAGGCC TATGAGGGGr TCCGCTATTA

GTGACATAT(

GCACTGCCCT

CCAACCGT'

CGCTGAGATT

GTGGCTCGTC

GTAAAGGTCT

CCCAGGTCGI

CCCTTCTCGG

TAATAAAACC

CGGGGCGGTT

TTAGAGACTA

CTCTCTTTTG

ATGCCAGTCA

CTTTCAGTCT

CACCTATGCC

GGTGCGCTAT

GTCGCCGCCG

TTTGCCCCCG

GCGTTTCACC

TCACCGCCTT

CTGTTCTGCC

GGCCCAGCGC

CTTTCGCTQA

CCTGAGGGGC

TCCTTGGCCC

GGCATGTTTG

GGAGTCGGCT

CACACTTTAC

ACCCGCACTT

CCTAGTCCAG

CCCAGCCCGA

CTTCTATACC

TAGTAGGGCC

CCCTCTACCC

CCCCCTGCAC

TCTGCTCCGG

CGCGTGGTGA

CCAGGGCCCC

AGCCATAACC

CCGCCTGCTC

TTTACCTACC

GCCGGCTCGC

TGTTTGAGTC

ACGCGTCTAA

TGTTGACCAC

TCATGCATTT

TACCAGAGGT

GCCTCTTTTG

TGATGCGCGA

TAACCCCCCG

GCCAATAATT

TAGGTTGGAA

CATAACCCAA

CGGGAGACTT

GCTCCCGCCT

TCCTCGGGAC

CGGCATATAC

r CTCGGCCGGC r GATGAGTCGG lk AGGCGGTCTC 3 CCAGGAGTGC 7 GTTGGCGACC P' CTGATGTTGA

"TGGGTCCTAC

TACCAAGCCC

CAGGCCGGCT

CGGGCGGATC

TTCCGCACGT

LATGGCCCAGA

GAGCACTATG

IGCCTCTGCTG

GGCTTGACCA

CCGGTCAGCC

CTATACAGGT

CCGGTAATTT

CTTTGCCCCG

AATCCATTCTC

GGTCGGAGGT

CTTAGGTTTT

I

GCCACACCTA

C

CGGATCCTTC

C

GCCGGCTCCT

C

CACCAGACGG C CGGATGGCTC GACATGTACC CGCCCTGGCG

G

ACCCCGCCTC

C

CGGCGCGGCC

G

CATGCCGTCG

C

AGAGGCTTGA

G

CGGTACCGCT

G

CAGGTGCCGA T

TTTCATCTTG

CCCCCTGCCA

AAAGTTTTGC

ACCTGTTTrC

AGGGCCATGA

CCCTGCTGAA

GTAGTCCCTG

TTGACCTCCC

GACCGCCACA

GATTGTGAGA

CGTTTGTTGA

GCGCCACAAT

GCCGCCGGCC

CTGGGCTGGA

CCGGGCGGTT

CCTGGCGAGG

rTAATCGCGA rTGGTTCCAT rTTTCCCCCG 3xTGGCGAGAG

[GATGCTGTT

~CATCTGAGC

~CCCGGCGGC

~CCTACTCTC

~GCGCTACCG

~CCGGCATCG

'AAGGTGTTC

'GGCTCGTTA

TGGGCTCTG

'TTTGATGCT

CCTACACAT

TCCTGATTA

GCTGCCTAC

CATACCCAC

CGGTCCCAG

1320 1360 1400 1440 1480 1520 1560 1600 1640 1680 1720 1760 1800 1840 1880 1920 1960 2000 2040 2080 2120 2160 2200 2240 2280 2320 2360 2400 2440 2480 2520 2560 2600 2640 2680 2720 WO 96/10580 PCTIIJS95/13 102 1.8 TTTTGACGCC TGGGAGCGGA ATCACCGCCC

CGGGGACGAG

TTGTACCTTC CTGAGCTTGC ATAGGCCGAC

CTGCCCAACT

TGCGCGGACA

GCAAATCTGG

ACAGACGTCG

GCCGGGCCTG

CCGGCGTTGT

GCAGTACCAG

ATCCGGCAAG

TCCCGCTCTA

GTTGTCGTGG

TCCCGACCCG

GCCGCCGCGG

CTTCGCTGCT

TAGAGTCACC

CAGGGGCGCC

CCGTCCCTTC

CCCCTCATTT

GGGCCGCCAC

CGTCCACCTT

CCCAGCCATC

GATTTTGAGC

ATCAGGCCCG

ATTTGGCCCC

CCCATCGCTG

CCCTGCGGAT

CGCATACCCT

ATGATTCAGA

TCGTTGTTCT

GGGGTGAGCC

TGTTCACCCA

GGCGGCTAAG

GACGGTCCAT

GAGGCACAGG

ACCATCATTG

CCACGGCAGA

CGTCCCGAGC

TCATGCCATT

TGAGAAGTGC

GTCATCATTG

GAGGTGGGCA

TCTCCGATGC.

TTGCTGGTGG

CGAAATTGGC

CCCTCGCGGC

AATCCTGACG

GCCTTCCCGC

CGTCTTGCCA

TGGCTGAGGA

GCTTGGCCAC

TGTTCTACCG CCCTGCCCTG TACCTGCCCC

AAGAACTCAC

CATTTGAATT AACAGATATT C CCCGAGCCAG CGCAAGGCCG CGTTATGGCC GCCGCACAAA

C

CTGATGTTCG CGACTCTCTC C TGGCCCCGTA CAGGTTACAA

C

GTGGAGGCCA

TGGTCGAGAAC

TGCCAGATGG

CTCACTATAA

CTATCGAACT

TGCCGGCTGT

TTTACCGCAG

TTACCCAAGC

GGAGTTGCGT

TTCACCCCGC

GGGTTGTCAT

GCTGCTGCTC

CTTGGCGACC

ACGCCGGGCT

CACCTCCTGG

GTATGTGAGC

CCACTAGTCG

CGCCGTTGGG

GCCGCCAACC

GCGCTACCTA

TGCTCGAGGC

GTTGCCTTGA

ACGCACCAGG

AATCGTTAAT

CACCAGCGCC

CCAATGTTGA

7ATTAGCGCC kGACCTGCCCC kGCTTGAACAC .ACCTGTGAT TGCATTGTC

C

7GCTGTCCAC

C

CTCTACAAT

G

CCCGTTTTA

TI

~CTGTGAATT

G

~GGCCAGGACG

TTCGAGGCCA

CTGAGGATGT

TGACTCAGCC

CGAGTCACCC

GTGTGCCTGG

CGACGTGGAC

AATGCCTGGC

ACACTGCGGC

TGATGAGGCC

CACATGCAGC

CGAATCAGAT

CGTTCCCGCC

TGGCATGTTA

TAATCCGCGG

GGTCCTCCGG

CAGAAGCTAG

CCGGTTCAGT

CACAGAGACT

CTCATTCAGT

CGCGCCACAC

CCTGCTTCGC

ACTTTTTCC

MATCTGTTAT

MACCTTGGCT

ETCCATCAGT

~TGTCGCGGC

;GGCCTTCTC

LGTGTCGTAA

TATGGCCGC

~CTCGTGGGC

~CCTCCCACT

'CCCGGCCAT

TACGAGCTA

*GCTCCGCCG

2760 2800 2840 2880 2960 3000 3040 3080 3120 3160 3200 3240 3280 3320 3360 3400 3440 3480 3520 3560 3600 3640 3680 3720 3760 3800 3840 3880 3920 3960 4000 4040 4080 4120 4160 TCCTTGAGCT CGACCTTTGT AGCCGCGACG

TGTCCAGGAT

m m WO 96/10580 PCTIUS95/13102 19

CACCTTCTTC

GAGACCATCG

CCTGGAGTAA

CCGTGCTATT

GGTGTGTTTTl

CGGCGGCTGT

TCTGAGTTTG

TAGAGTGTGC

GCTCATCCGC

CTGCAGGCCC

AACACTCCGG

CTGGAACATG

GATCTGCAGG

TGCTTTGCAG

CCTGATTGCT

CGTCCGATTG

GCCTTGGCGC

GCTTACTGAG

GAGCAGCTCC

TCACGAATGT

TGTTTATGGG

GGCATGCTAC

CTGAGTCAGTC

TCTGTGTCGG

C

CCATGGGTTC C TTGCTCCTCA CCGGTCAGCC

C

CGGTTCCGGC

C

CAGCCCTTCGC

TCGCCCCCGA TGTTCGCCAA

C

GACCAGGCCCA

CTACCACAGCT

GGCCCATGAC A GGCGCCATCC

T

CCCTCACCTC

T

CAGAAAGAT

CCCATGGTA

GACCTTCTG'

GAGAAGGCTZ

ATGGGGATGi GGCCGCAGCj

ATTCCACCCJ

TATTATGGA(

TTGTACCACC

CGAAGGAGT(

TGAGCCCGGC

GCCGTTATCI

TGGCTGCCTI

TGAGTACCGI

GGCTGTGGCC

GTCTGTATGC

GCTTCCTGAT

AAGAATTGGG

3CCTCGCTGT

.,GCTCAGATG

3TTTCCCCTG kGGCTGTTGC

'AAGCCAGTG

TGGAATGAA

CGACCATGC

'GTTTCTGCC

~TCTGGCCGC

~GTGGTTTCT

!AATCCCCTA

'GTCACCGCT

CCGCCCGAC

.GCGCCCCGC

'GGGGCCGCG

CCCCGCCAG

GCGCCGGCA

T GTAATAAATT A AGTGGGCCAG F GCCCTTTTCG k~ TCCTGGCCCT :7 CTTTGATGAC k AAGGCATCCA k GAATAATTTT 3GAGTGTGGGA 7TTATAAGGTC

SCCTGCGAGGG

7ACCCTTCTGT

LCCCACTGTTA

'TAAAGGTGAT

'CAGAGCCCAG

TAAAGTTGAA

AGGTGTTGTG

GTCGTGCGCT

GCCCTGGCCC

GAGTGATTTT

TGTGTGGATG

GGCTCGTTCA

TGATGGCAAG

CTTGACCTGA

TAACATGTCT

GCCCTCGGCC

TATGCTGCCCC

CGTCGTGGGC

C

GGGGTGACCG

TATTCATCCA 7 GCGGCCGOGG

C

CACTCGGCTC

C

CGCTGCCTCA

C

CCGCTAACCG

C

TGCCTGATGT GTATAACCTA TI

CACCACGGGG

GGCATTTCGG

GCCCCTGGTT

GCTCCCTCAG

ACCGTCTTCT

GAATGACTTT

TCCTTGGGCC

TGCCGCAGTG

TGCGTGGATT

TTTTGGAAGA

GGAATACTGT

TGATTTCCGC

GATTCGATAG

GGGCTGCTGT

GGTGGATTTC

GTGGCCCCCG

TCGCCGGTCG

CGAGCGGGCG

CTCCGCAAGC

TTGTCTCTCG

rAACCTGATT .7CTCATTTCA

CAAATTCAAT

E'TTGCTGCGC

['ATTTTGCTG

;CGCCACCGC

GCGCAGCGG

;GTTGATTCT

~CCAACCCCT

~TGGACCTCG

~GCTTGGCGT

GTCGTAGAC

'GGTCGCTCC

'GACTCCCGC

'CAACATCTC

4200 4240 4280 4320 4360 4400 4440 4480 4520 4560 4600 4640 4680 4720 4760 4800 4840 4880 4920 4960 5000 5040 5080 5120 5160 5200 5240 5280 5320 5360 5400 5440 5480 5520 5560 5600 TCCGTGGCC ACCGGCACAA

ATTTGGTTCT

WO 96/10580 13102 20 TTACGCCGCT CCTCTTAGC( GGCACCAATA CTCATATAA ATGCCCAGTA CCGGGTTGC': CCCGCTGGTC CCCAACGCTC ATTTCGTTCT GGCCACAGAC TTGACATGAA TTCAATAAC( AGTCCAGCCC GGCATAGCC9 GAGCGCCTAC ACTATCGCAI AGACCTCCGG GGTGGCGGAC TGTCATGCTC

TGCATACATC

ACTAATACAC

CCTATACCGC

TTGCCCTCGA ACTTGAGTTC TACCAATACG

CGGGTCTCGC

CACCGCCTTC GTCGCGGTGC CCACCACGGC TGCTACTCGC TACTAGTACT

AATGGTGTTG

GCGCTTACCC

TGTTTAACCT

GTCTACCGAC

AGAATTGATT

GTTCTACTCT

CGCCCCGTCG

ACTGTTAAGC TGTATACATC ATAAGGGTAT

TGCAATCCCG

ATCCCGTGTA GTTATTCAGG CAGGACCGAC CGACACCTTC TTTCTGTCCT CCGAGCTAAC CACCGCTGCC GAGTATGACC ACCGGCCCAG

TCTATGTCTC

ATGTTGCGAC CGGCGCGCAG CTGGACCAAG GTCACACTTG ATCCAGCAGT ATTCAAAGAC GCGGTAAGCT

CTCCTTTTGG

CGGGTACCCT

TATAATTATA

CTGCTCGTTG AGAATGCCGC CCACCTACAC TACTAGCCTG TTCCGCGGTT GCTGTTTTAG TTGCTTGAGG ATACCATGGA CTTTCGATGA

CTTCTGCCCG

7CGCTTCTACC r' GGcTAcAGAA r CGTGCCACAA

TTGGTGGCTA

SCACCACCACC

STCGACGGATG

7 CCGAGCTTGT k CCAAGGTTGG

GAGGAGGCCA

GCTCACCTGT

TGCCCTCGGG

CGCAACCTCA

*GTTACTCCAG

*AGATGGGACT

ITTCATGAAGG

GTGAGATCGG

TGCTGACACC

TCGTCGGCTG

TCTCAGCCAA

TGTGGAGAAT

CATGACATCG.

ATTATGACAA

CCCAGCCCCA

GATGTGCTTT

AGTCCACTTA

TGACTCTGTG

GCCGTTGCCC

ATGGTCGCCC

CTTCTTTGTC

GAGGCAGGAA

ACACCACTGC TGGGCATCGG C GGTGCTGGCC C CCCCCCACTC CTACCCTGCc C GAGTGCCGCC C

CCTCCAGGAC

GCTTCTAATT

TTCGCTACCG

CGCTATCTCC

CCGACGTCCG

TCCGTATTTT

TATTCCAAGT

CGCTCTGTTG

CCTCTGGTCT

AAATTCTTAT

CTGTTGGACT

CCCCCGGTAA

CACTGCCCGT

GCCGAGCTCA

ACCTCTATTT

CCGCGGGATA

CTGCTTGGCG

GTGGCCAGCT

TGGCGAGCCG

GCTCAGCAGG

ACCTCGGGGA

CCAACATGAG

rCGCGTCCTT 3GCTTTCTCT

CGGCTCTTCG

kCCTTGGTTA 3GTCACTCGA

XCTTTCCACC

7TGCCGCTCC

~TACTAAAGC

L AGTGACCAA

~TTGCTATTT

~CGTCTCTAT

.'GTGCTAGCA

GCGCCCATA

~CCTTGGCCT

5640 5680 5720 5760 5800 5840 5880 5920 5960 6000 6040 6080 6120 6160 6200 6240 6280 6320 6360 6400 6440 6480 6520 6560 6600 6640 6680 6720 6760 6800 6840 6880 6920 6960 7000 7040 WO 96/10580 PCT/US95/13102 -21 0 CCAGGGTTGT GCTTTTCAGT CTACTGTCGC TGAGCTTCAG 7080 CGCCTTAAGA TGAAGGTGGG TAAAACTCGG GAGTTATAGT 7120 TTATTTGCTT GTGCCCCCCT TCTTTCTGTT GCTTATTT 7168 The abbreviations used for the nucleotides are those standardly used in the art.

The sequence in one direction has been designated by convention as the "plus" sequence since it is the protein-encoding strand of RNA viruses and this is the sequence shown above as SEQ ID. NO.:4.

The deduced amino acid sequences of the open reading frames of SAR-55 have SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3. ORF-1 starts at nucleotide 28 of SEQ.

ID NO. 4 and extends 5078 nucleotides; ORF-2 starts at nucleotide 5147 of SEQ. ID NO. 4 and extends 1979 nucleotides; and ORF-3 starts at nucleotide 5106 of SEQ. ID NO.

4 and extends 368 nucleotides.

Variations are contemplated in the DNA sequence S which will result in a DNA sequence that is capable of 2 directing production of analogs of the ORF-2 protein. By "analogs of the ORF-2 protein" as used throughout the specification and claims is meant a protein having an amino acid sequence substantially identical to a sequence specifically shown herein where one or more of the residues shown in the sequences presented herein have been substituted with a biologically equivalent residue such that the resultant protein the "analog") is capable of forming viral particles and is immunogenic. It should be noted that the DNA sequende set forth above represents 3 a preferred embodiment of the present invention. Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a DNA sequence capable of directing production of the instant ORF proteins or their analogs. As such, DNA sequences which are functionally equivalent to the WO 96/10580 PCT/US95/13102 22 0 sequences set forth above or which are functionally equivalent to sequences that would direct production of analogs of the ORF proteins produced pursuant to the amino acid sequence set forth above, are intended to be encompassed within the present invention.

The present invention relates to a method for detecting the hepatitis E virus in biological samples based on selective amplification of hepatitis E gene fragments. Preferably, this method utilizes a pair of single-stranded primers derived from non-homologous regions of opposite strands of a DNA duplex fragment, which in turn is derived from a hepatitis E virus whose genome contains a region homologous to the SAR-55 sequence shown in SEQ ID No.: 4. These primers can be used in a 1 method following the process for amplifying selected nucleic acid sequences as defined in U.S. Patent No.

4,683,202.

The present invention also relates to the use of single-stranded antisense poly-or oligonucleotides derived from sequences homologous to the SAR-55 cDNA to inhibit 2 the expression of hepatitis E genes. These anti-sense poly-or oligonucleotides can be either DNA or RNA. The targeted sequence is typically messenger RNA and more preferably, a signal sequence required for processing or translation of the RNA. The antisense poly-or oligonucleotides can be conjugated to a polycation such as polylysine as disclosed in Lemaitre, M. et al. (1989) Proc Natl Acad Sci USA 84:648-652; and this conjugate can be administered to a mammal in an amount sufficient to hybridize to and inhibit the function of the messenger

RNA.

The present invention includes a recombinant

DNA

method for the manufacture of HEV proteins, preferably a protein composed of at least one ORF protein, most preferably at least one ORF-2 protein. The recombinant 3 ORF protein may be composed of one ORF protein or a WO 96/10580 PCTUS95/13102 23 0 combination of the same or different ORF proteins. A natural or synthetic nucleic acid sequence may be used to direct production of the HEV proteins. In one embodiment of the invention, the method comprises: preparation of a nucleic acid sequence capable of directing a host organism to produce a protein of HEV; cloning the nucleic acid sequence into a vector capable of being transferred into and replicated in a host organism, such vector containing operational elements for the nucleic acid sequence; transferring the vector containing the nucleic acid and operational elements into a host organism capable of expressing the protein; culturing the host organism under conditions appropriate for amplification of the vector and expression of the protein; and harvesting the protein.

In another embodiment of the invention, the method for the recombinant DNA synthesis of a protein encoded by nucleic acids of HEV, preferably encoding by at least one ORF of HEV or a combination of the same or different ORF proteins, most preferably encoding at least one ORF-2 nucleic acid sequence, comprises: culturing a transformed or transfected host organism containing a nucleic acid sequence capable of directing the host organism to produce a protein, under conditions such that the protein is produced, said protein exhibiting substantial homology to a native HEV protein isolated-from HEV having the amino acid sequence according to SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, or combinations thereof.

In one embodiment, the RNA sequence of the viral genome of HEV strain SAR-55 was isolated and cloned to cDNA as follows. Viral RNA is extracted from a biological sample collected from cynomolgus monkeys infected with WO 96/10580 PCT/US95/13102 24 0 and the viral RNA is then reverse transcribed and amplified by polymerase chain reaction using primers complementary to the plus or minus strands of the genome of a strain of HEV from Burma (Tam et al. (1991)) or the genome. The PCR fragments are subcloned into pBR322 or pGEM-32 and the double-stranded PCR fragments were sequenced.

The vectors contemplated for use in the present invention include any vectors into which a nucleic acid sequence as described above can be inserted, along with any preferred or required operational elements, and which vector can then be subsequently transferred into a host organism and replicated in such organism. Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements preferred or required for transcription of the nucleic acid sequence.

The "operational elements" as discussed herein include at least one promoter, at least one operator, at least one leader sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector nucleic acid. In particular, it is contemplated that such vectors will contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence.

In construction of the cloning vector of the present invention, it should additionally be noted that multiple copies of the nucleic acid sequence and its attendant operational elements may be inserted into each vector. In such an embodiment, the host organism would produce greater amounts per vector of the desired

HEV

protein. The number of multiple copies of the DNA sequence which may be inserted into the vector is limited only by the ability of the resultant vector due to its size, to be transferred into and replicated and transcribed in an appropriate host microorganism.

In another embodiment, restriction digest fragments containing a coding sequence for HEV proteins can be inserted into a suitable expression vector that functions in prokaryotic or eukaryotic cells. By suitable is meant that the vector is capable of carrying and expressing a complete nucleic acid sequence coding for HEV proteins, preferably at least one complete ORF protein. In the case or ORF-2, the expressed protein should form viral-like particles. Preferred expression vectors are those that function in a eukaryotic cell. Examples of such vectors include but are not limited to vectors useful for expression in yeast pPIC9 vector-lnvitrogen) vaccinia virus vectors, adenovirus or herpesviruses, preferably the baculovirus transfer vector, pBlueBac. Preferred vectors are p63-2, which contains the complete ORF-2 gene, and P59-4, which contains the complete ORF-3 and ORF-2 genes. These vectors were 15 deposited with the American Type Culture Collection, 12301 Parklawn Drive, S. Rockville, MD 20852 USA on September 9, 1992 and have accession numbers 75299 (P63-2) and 75300 (P59-4). Example 1 illustrates the cloning of the ORF-2 gene into pBlueBac to produce p63-2. This method includes digesting the genome of HEV strain SAR-55 with the restriction enzymes Nrul and Bglll, 20 inserting a polylinker containing B1nl and Bglll sites into the unique Nhel site of the vector and inserting the Nrul-Bglll ORF-2 fragment in Blnl-Bglll PBlueBac using an adapter.

In yet another embodiment, the selected recombinant expression vector may then be transfected into a suitable eukaryotic cell system for purposes of 25 expressing the recombinant protein. Such eukaryotic cell systems include, but are not limited to, yeast, and cell lines such as HeLa, MRC-5 or Cv-1. One preferred

SEC

S104 Q, /VnT O' WO 96/10580 PCTIUS95/13102 26 0 eukaryotic cell system is SF9 insect cells. One preferred method involves use of the pBlueBac expression vector where the insect cell line SF9 is cotransfected with recombinant pBlueBac and AcMNPV baculovirus DNA by the Ca precipitation method.

The expressed recombinant protein may be detected by methods known in the art which include Coomassie blue staining and Western blotting using sera containing anti-HEV antibody as shown in Example 2.

1 Another method is the detection of virus-like particles by immunoelectron microscopy as shown in Example 3.

In a further embodiment, the recombinant protein expressed by the SF9 cells can be obtained as a crude lysate or it can be purified by standard protein purification procedures known in the art which may include 1 differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity, and immunoaffinity chromatography and the like. In the case of immunoaffinity chromatography, the recombinant protein 2 may be purified by passage through a column containing a resin which has bound thereto antibodies specific for the ORF protein. An example of a protocol for the purification of a recombinantly expressed HEV ORF protein is provided in Example In another embodiment, the expressed recombinant proteins of this invention can be used in immunoassays for diagnosing or prognosing hepatitis E in a mammal including but not limited to humans, chimpanzees, Old World monkeys, New World monkeys, other primates and the like. In a 3 preferred embodiment, the immunoassay is useful in diagnosing hepatitis E infection in humans. Immunoassays using the HEV proteins, particularly the ORF proteins, and especially ORF 2 proteins, provide a highly specific, sensitive and reproducible method for diagnosing

HEV

infections, in contrast to immunoassays which utilize WO 96/10580 PCTfUS9/13102 27 0 partial ORF proteins. Immunoassays of the present invention may be a radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like. Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbell et al., Methods of Immunology, W.A. Benjamin, Inc., 1964, both of which are incorporated herein by reference. Such 1 assays may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art.

(Oellerich, M. 1984. J.Clin. Chem. Clin. BioChem. 22: 895-904) Biological samples appropriate for such detection assays include, but are not limited to, tissue biopsy extracts, whole blood, plasma, serum, cerebrospinal 1 fluid, pleural fluid, urine and the like.

In one embodiment, test serum is reacted with a solid phase reagent having surface-bound recombinant HEV protein as an antigen, preferably an ORF protein or combination of different ORF proteins such as ORF-2 and 2 ORF-3, ORF-1 and ORF-3 and the like. More preferably, the HEV protein is an ORF-2 protein that forms virus-like particles. The solid surface reagent can be prepared by known techniques for attaching protein to solid support material. These attachment methods include non-specific 2 adsorption of the protein to the support or covalent attachment of the protein to a reactive group on the support. After reaction of the antigen with anti-HEV antibody, unbound serum components are removed by washing and the antigen-antibody complex is reacted with a secondary antibody such as labelled anti-human antibody.

The label may be an enzyme which is detected by incubating the solid support in the presence of a suitable fluorimetric or colorimetric reagent. Other detectable labels may also be used, such as radiolabels or colloidal gold, and the like.

1 WO 96/10580 PCT/US95/13102 28 0 In a preferred embodiment, protein expressed by the recombinant vector pBlueBac containing the entire ORF- 2 sequence of SAR-55 is used as a specific binding agent to detect anti-HEV antibodies, preferably IgG or IgM antibodies. Examples 4 and 5 show the results of an ELISA in which the solid phase reagent has recombinant ORF-2 as the surface antigen. This protein, encoded by the entire ORF-2 nucleic acid sequence, is superior to the partial ORF-2 proteins, as it is reactive with more antisera from different primate species infected with HEV than are partial antigens of ORF-2. The protein of the present invention is also capable of detecting antibodies produced in response to different strains of HEV but does not detect antibodies produced in response to Hepatitis A, B, C or Hepatitis D.

The HEV protein and analogs may be prepared in the form of a kit, alone, or in combinations with other reagents such as secondary antibodies, for use in immunoassays.

The recombinant HEV proteins, preferably an ORF protein or combination of ORF proteins, more preferably an ORF-2 protein and substantially homologous proteins and analogs of the invention can be used as a vaccine to protect mammals against challenge with Hepatitis E. The vaccine, which acts as an immunogen, may be a cell, cell 2 lysate from cells transfected with a recombinant expression vector or a culture supernatant containing the expressed protein. Alternatively, the immunogen is a partially or substantially purified recombinant protein.

While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.

The formulations of the present invention, both for veterinary and for human use, comprise an immunogen as described above, together with one or more WO 96/10580 PCT/US95/13102 29 0 pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art.

All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which 20 are preferably isotonic with the blood of the recipient.

Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride 0.1-2.0M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. These may be present in unit or multi-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 M

_M

WO 96/10580 PCTIUS95/13102 30 0 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH. The specific osmotic pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8- 1.2. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. In formulating the immunogen of the present invention, anti-adsorption agent may be used.

Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or absorb the proteins or their derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyester, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled-release preparations is to incorporate the proteins, protein analogs or their functional derivatives, 2 into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules 3 prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and WO 96/10580 PCT/US95/13102 31 0 nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.

The proteins of the present invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above.

Vaccination can be conducted by conventional methods. For example, the immunogen can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. Further, the immunogen may or may not be bound to a carrier to make the protein immunogenic.

1 Examples of such carrier molecules include but are not limited to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like. The immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, 2 intramuscular, subcutaneous, and the like. The immunogen may be administered once or at periodic intervals until a significant titer of anti-HEV antibody is produced. The antibody may be detected in the serum using an immunoassay.

25 In yet another embodiment, the immunogen may be nucleic acid sequence capable of directing host organism synthesis of an HEV ORF protein. Such nucleic acid sequence may be inserted into a suitable expression vector by methods known to those skilled in the art. Expression vectors suitable for producing high efficiency gene transfer in vivo include, but are not limited to, retroviral, adenoviral and vaccinia viral vectors.

Operational elements of such expression vectors are disclosed previously in the present specification and are known to one skilled in the art. Such expression vectors WO 96/10580 PCT/US95/13102 32 0 can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or orally.

In an alternative embodiment, direct gene transfer may be accomplished via intramuscular injection of, for example, plasmid-based eukaryotic expression vectors containing a nucleic acid sequence capable of directing host organism synthesis HEC ORF protein(s).

Such an approach has previously been utilized to produce the hepatitis B surface antigen in vivo and resulted in an antibody response to the surface antigen (Davis, H.L. et al. (1993) Human Molecular Genetics, 2:1847-1851; see also Davis et al. (1993) Human Gene Therapy, 4:151-159 and 733- 740).

When the immunogen is a partially or substantially purified recombinant HEV ORF protein, 1 dosages effective to elicit a protective antibody response against HEV range from about 2 pg to about 100 Ag. A more preferred range is from about 5 pg to about 70 Ag and a most preferred range is from about 10 pg to about 60 pg.

Dosages of HEV-ORF protein encoding nucleic 2 acid sequence effective to elicit a protective antibody response against HEV range from about 1 to about 5000 pg; a more preferred range being about 300 to about 1000 pg.

The expression vectors containing a nucleic acid sequence capable of directing host organism synthesis of 2 an HEV ORF protein(s) may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above.

The administration of the immunogen of the present invention may be for either a prophylactic or 3 therapeutic purpose. When provided prophylactically, the immunogen is provided in advance of any exposure to HEV or in advance of any symptom due to HEV infection. The prophylactic administration of the immunogen serves to prevent or attenuate any subsequent infection of HEV in a 3 mammal. When provided therapeutically, the immunogen is WO 96/10580 PCT/US95/13102 33 provided at (or shortly after) the onset of the infection or at the onset of any symptom of infection or disease caused by HEV. The therapeutic administration of the immunogen serves to attenuate the infection or disease.

A preferred embodiment is a vaccine prepared using recombinant ORF-2 protein expressed by the ORF-2 sequence of HEV strain SAR-55 and equivalents thereof.

Since the recombinant ORF-2 protein has already been demonstrated to be reactive with a variety of HEV-positive sera, their utility in protecting against a variety of HEV strains is indicated.

In addition to use as a vaccine, the compositions can be used to prepare antibodies to HEV virus-like particles. The antibodies can be used directly as antiviral agents. To prepare antibodies, a host animal is immunized using the virus particles or, as appropriate, non-particle antigens native to the virus particle are bound to a carrier as described above for vaccines. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the virus particle. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs.

The antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas. Humanized antibodies nonimmunogenic in a human) may be produced, for example, by replacing an immunogenic portion of an antibody with a WO 96/10580 WO 96/10580 PCTIUS95/13102 -34 corresponding, but nonimmunogenic portion chimeric antibodies). Such chimeric antibodies may contain the reactive or antigen binding portion of an antibody from one species and the Fc portion of an antibody (nonimmunogenic) from a different species. Examples of chimeric antibodies, include but are not limited to, nonhuman mammal-human chimeras, rodent-human chimeras, murine-human and rat-human chimeras (Robinson et al., International Patent Application 184,187; Taniguchi

M.,

European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al., 1987 Proc. Natl.

Acad. Sci. USA 84:3439; Nishimura et al., 1987 Canc. Res.

47:999; Wood et al., 1985 Nature 314:446; Shaw et al., S 1988 J. Natl. Cancer Inst. 80: 15553, all incorporated herein by reference).

General reviews of "humanized" chimeric antibodies are provided by Morrison 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques 4:214.

Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan et al., 1988 Science 239:1534; Biedleret al. 1988 J. Immunol. 141:4053, all incorporated herein by reference).

The antibodies or antigen binding fragments may 2 also be produced by genetic engineering. The technology for expression of both heavy and light cain genes in E.

coli is the subject the PCT patent applications; publication number WO 901443, W0901443, and WO 9014424 and in Huse et al., 1989 Science 246:1275-1281.

The antibodies can also be used as a means of enhancing the immune response. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb 3 body weight during the early incubation period of other WO 96/10580 PCT/US95/13102 0 viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the HEV virus particle can be passively administered alone or in conjunction with another antiviral agent to a host infected with an HEV to enhance the effectiveness of an antiviral drug.

Alternatively, anti-HEV antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-HEV antibody preparation prepared as described above is used to induce 0 anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent.

Following administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies produced 1 by the same species as the host animal can be used or the FC region of the administered antibodies can be removed.

Following induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody composition. The composition can be purified as described 2 above for anti-HEV antibodies, or by affinity chromatography using anti-HEV antibodies bound to the affinity matrix. The anti-idiotype antibodies produced are similar in conformation to the authentic HEV-antigen and may be used to prepare an HEV vaccine rather than using an HEV 2 particle antigen.

When used as a means of inducing anti-HEV virus antibodies in an animal, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desirable.

The HEV derived proteins of the invention are also intended for use in producing antiserum designed for pre- or post-exposure prophylaxis. Here an HEV protein, WO 96/10580 PCT/US95I13102 36 or mixture of proteins is formulated with a suitable adjuvant and administered by injection to human volunteers, according to known methods for producing human antisera. Antibody response to the injected proteins is monitored, during a several-week period following immunization, by periodic serum sampling to detect the presence of anti-HEV serum antibodies, using an immunoassay as described herein.

The antiserum from immunized individuals may be administered as a pre-exposure prophylactic measure for individuals who are at risk of contracting infection. The antiserum is also useful in treating an individual postexposure, analogous to the use of high titer antiserum against hepatitis B virus for post-exposure prophylaxis.

Of course, those of skill in the art would readily understand that immune globulin (HEV immune globulin) purified from the antiserum of immunized individuals using standard techniques may be used as a pre-exposure prophylactic measure or in treating individuals postexposure.

20 For both in vivo use of antibodies to HEV viruslike particles and proteins and anti-idiotype antibodies and diagnostic use, it may be preferable to use monoclonal antibodies. Monoclonal anti-virus particle antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. (Goding, J.W. 1983.

Monoclonal Antibodies: Principles and Practice, Pladermic Press, Inc., NY, NY, pp. 56-97). To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with HEV (where infection has been shown for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the WO 96/10580 PCTIUS95/13102 37 0 donor is subject to splenectomy. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.

Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. For monoclonal anti-virus particle antibodies, the antibodies must bind to HEV virus particles. For monoclonal anti-idiotype antibodies, the antibodies must bind to anti-virus particle antibodies. Cells producing antibodies of the desired specificity are selected.

The above described antibodies and antigen 1 binding fragments thereof may be supplied in kit form alone, or as a pharmaceutical composition for in vivo use.

The antibodies may be used for therapeutic uses, diagnostic use in immunoassays or as an immunoaffinity agent to purify ORF proteins as described herein.

Material The materials used in the Examples were as follows: Primates. Chimpanzee (Chimp) (Pan troglodytes).

Old world monkeys: cynomolgus monkeys (Cyno) (Macaca 2 fascicularis), rhesus monkeys (Rhesus) mulatta), pigtail monkeys (PT) nemestrina), and African green monkeys (AGM) (Cercopithecus aethiops). New World monkeys: mustached tamarins (Tam) (Saguinus mystax), squirrel monkeys (SQM) (Saimiri sciureus) and owl monkeys (OWL) (Aotus trivigatus). Primates were housed singly under conditions of biohazard containment. The housing, maintenance and care of the animals met or exceeded all requirements for primate husbandry.

Most animals were inoculated intravenously with HEV, strain SAR-55 contained in 0.5 ml of stool suspension WO 96/10580 PCT/US95/13102 -38 0 diluted in fetal calf serum as described in Tsarev, S.A.

et al. (1992), Proc. Natl. Acad. Sci USA, 89:559-563; and Tsarev, S.A. et al. (1993), J. Infect. Dis. (167:1302- 1306). Chimp-1313 and 1310 were inoculated with a pool of stools collected from 7 Pakistani hepatitis E patients.

Serum samples were collected approximately twice a week before and after inoculation. Levels of the liver enzymes serum alanine amino transferase (ALT), isocitrate dehydrogenase (ICD), and gamma glutamyl transferase (GGT) were assayed with commercially available tests (Medpath Inc., Rockville, MD). Serologic tests were performed as described above.

EXAMPLE 1 Identification of the DNA Sequence of the Genome of HEV Strain Preparation of Virus RNA Template for PCR. Bile from an HEV-infected cynomolgus monkey (10 al), (wt/vol) SDS (to a final concentration of proteinase K (10 mg/ml; to a final concentration of 1 mg/ml), 1 Al of tRNA (10 mg/ml), and 3 tl of 0.5 M EDTA were mixed in a final volume of 250 tl and incubated for 30 min. at 55 0

C.

Total nucleic acids were extracted from bile twice with phenol/chloroform, 1:1 (vol/vol), at 65 0 C and once with chloroform, then precipitated by ethanol, washed with ethanol, and used for RT-PCR. RT-PCR amplification of HEV RNA from feces and especially from sera was more efficient when RNA was more extensively purified. Serum (100 Al) or a 10% fecal suspension (200 pl) was treated as above with proteinase K. After a 30-min incubation, 300 pl of CHAOS buffer (4.2 M guanidine thiocyanate/0.5

N-

lauroylsarocosine/0.025 M Tris-HCL, pH 8.0) was added.

Nucleic acids were extracted twice with phenol/chloroform at 65 0 C followed by chloroform extraction at room temperature. Then 7.5 M ammonium acetate (225 Al) was added to the upper phase and nucleic acids were precipitated with 0.68 ml of 2-propanol. The pellet was WO 96/10580 PCTIUS95/13102 39 0 dissolved in 300 ul CHAOS buffer and 100 ul of H 2 0 was added. Chloroform extraction and 2-propanol precipitation were repeated. Nucleic acids were dissolved in water, precipitated with ethanol, washed with 95% ethanol, and used for RT-PCR.

Primers. Ninety-four primers, 21-40 nucleotides (nt) long, and complementary to plus or minus strands of the genome of a strain of HEV from Burma (BUR-121) (Tam, A.W. et al. (1991), Virology, 185:120-131) or the 1 genome were synthesized using an Applied Biosystems model 391 DNA synthesizer.

The sequences of these 94 primers are shown below starting with SEQ. ID NO. 5 and continuing to SEQ.

ID NO. 98: HEV Primer List Primer

D

R

D

R

D

R

3042 3043 3044 3045 261 260 259 255

ORF

Region 1 1 1 1 1 1 1 1 Sequence

ACATTTGAATTCACAGACAT

TGTGC

ACACAGATCTGAGCTACATT

CGTGAG

AAAGGGATCCATGGTGTTTG

AGAATGZ

ACTCACTGCAGAGCACTATC

GAATC

CGGTAAACTGGTACTGCACA

AC

AAGTCCCGCTCTATTACCCA

AG

ACCCACGGGTGTTGGTTTTT

G

TTCTTGGGGCAGGTAGAGAA

G

(SEQ.

(SEQ. 3 (SEQ. I

ID

ED.

[D.

:D.

NO.

6) 7) 8) 9)

LO)

L1) .2) R 254 S 2 TTATTGAATTCATGTCAACG

GACGTC

(SEQ. ID. NO. 13) WO 96/10580 PCTIUS95/13102 242 S

R

D

R

D

241 231 230 229 228 227 218 217 211 40

AATAATTCATGCCGTCGCTC

C

AAGCTCAGGAAGGTACAACT

c

AAATCGATGGCTGGGATCTG

ATTC

GAGGCATTGTAGAGCTTTGT

G

GATGTTGCACGGACAGCAAA

TC

ATCTCCGATGCAATCGTTAA

TAAC

TAATCCATTCTGTGGCGAGA

G

AAGTGTGACCTTGGTCCAGT

C

TTGCTCGTGCCACAATTCGC

TAC

CATTTCACTGAGTCAGTGAA

GZ

TAATTATAACACCACTGCTA

G

GATTGCAATACCCTTATCCT

G

ATTAAACCTGTATAGGGCAG

AAC

AAGTTCGATAGCCAGATTTG

C

TCATGTTGGTTGTCATAATC

C

GATGACGCACTTCTCAGTGT

G

AGAACAACGAACGGAGAAC

(SEQ. ID. NO. 14)

(SEQ.

ID.

NO.

16) 17) 18) 19) 21) 22) 23) 24) 26) 27) 28) 29)

D

R

202 201 200 199 198 193 192 D 191 B 1 AGATCCCAGCCATCGACTTT

G

(SEQ. ID. NO. 31) WO 96/10580 WO 9610580PCTfUS9S/13102 41 2 TAGTAGTGTAGGTGGAAATA

G

R 190 S (SEQ. ID. NO. 32)

D

R

D

R

D

R

D

R

189 188 187 186 185 184 181 180 179 178 177 174 173 172 166 143 2 2 2 2 2,3 2,3 2

GTGTGGTTATTCAGGATTAT

G

ACTCTGTGACCTTGGTTAAT

G

AACTCAAGTTCGAGGGCAAA

G

CGCTTACCCTGTTTAACCTT

G

ATCCCCTATATTCATCCAAC

CAAC

CTCCTCATGTTTCTGCCTAT

G

GCCAGAACGAAATGGAGATA

GC

CTCAGACATAAAACCTAAGT

C

TGCCCTATACAGGTTTAATC

G

ACCGGCATATACCAGGTGC

ACATGGCTCACTCGTAAATT

C

AACATTAGACGCGTTAACGA

G

CTCTTTTGATGCCAGTCAGA

G

ACCTACCCGGATGGCTCTAA

TATGGGAATTCGTGCCGTCC

TGAAG (EcoRI)

AGTGGGAGCAGTATACCAGC

G

(SEQ.

ID.

ID.'

ID.

*NO.

NO.

33) 34) 36) 37) 38) 39) 41) 42) 43) 44) 46) 47) (SEQ.*ID. NO. 48) D 141 B 1 CTGCTATTGAGCAGGCTGCT

C

(SEQ. ID.NO49 NO. 49) WO 96/10580 PCTIUS95/13102 42 R 142 S 1 GGGCCATTAGTCTCTAAAAC

D

R

D

R

D

R

135 134 133 132 131 119 118 117 2,3 5' NC

GAGGTTTTCTGGAATCATC

GCATAGGTGAGACTG

AGTTACAGCCAGAAAACC

CCATGGATCCTCGGCCTATT

TTGCTGTTGCTCC (Barn HI)

AGGCAGACCACATATGTG

GGTGCACTCCTGACCAAGCC

ATTGGCTGCCACTTTGTTC

ACCCTCATACGTCACCACAA

C

(SEQ.

ID.

NO.

51) 52) 53) 54) 56) 57) (SEQ. ID. NO. 58) R 116 B 1 GCGGTGGACCACATTAGGAT

TATC

D 115 B 1 CATGATATGTCACCATCTG D 114 B 1 GTCATCCATAACGAGCTGG R 112 B 2 AGCGGAATTCGAGGGGCGGC ATAAAGAACCAGG (EcoRI) R 111 B 2 GCGCTGAATTCGGATCACAA

GCTCAGAGGCTATGCC

(EcoRI) D 110 B 2 GTATAACGGATCCACATCTC CCCTTACCTC (Barn HI) D 109 B 2 TAACCTGGATCCTTATGCCG CCCCTCTTAG (Barn HI) D 108 B 1 AAATTGGATCCTGTGTCGGG

TGGAATGAATAACATGTC

(BainHI) R 107 B 1 ATCGGCAGATCTGATAGAGC

GGGGACTTGCCGGATCC

D 101 B 2 TACCCTGCCCGCGCCCATAC

TTTTGATG

R 100 B 1 GGCTGAGATCTGGTTCGGGT CGCCAAGAAGGTG (Bgl II)

(SEQ.

ID.

NO.

59) 61) (SEQ. ID. NO. 62) (SEQ. ID. NO. 63) (SEQ. ID. NO. 64) (SEQ. ID. NO. (SEQ. ID. NO. 66) (SEQ. ID. NO. 67) (SEQ. ID. NO. 68) (SEQ. ID. NO. 69) WO 96/10580 PCT/US95/13102

R

D

R

D

R

D 90 R 89 R 88 R 87 D 86 R 81 D 80 R 79 D 78 R 77 43 2 TACAGATCTATACAACTTAA CAGTCGG (Bg1 II) 2 GCGGCAGATCTCACCGACAC CATTAGTAC (Bgl II) 1 CCGTCGGATCCCAGGGGCTG CTGTCCTG (Barn HI) 2 AAAGGAATTCAAGACCAGAG GTAGCCTCCTC (EcoRI) 2 GTTGATATGAATTCAATAAC

CTCGACGG

3 'NC TTTGGATCCTCAGGGAGCGC

GGAACGCAGAAATGAG

(BainHI) 2 TCACTCGTGAATTCCTATAC TAATAC (EcoRI) 3' NC TTTGGATCCTCAGGGAGCGC GGAACGCAGAAATG (BainHI) 1 TGATAGAGCGGGACTTGCCG GATCC (BainHI) 1 TTGCATTAGGTTAATGAGGA

TCTC

1 ACCTGCTTCCTTCAGCCTGC

AGAAG

1 GCGGTGGATCCGCTCCCAGG CGTCAAAAC (BarnHI) 1 GGGCGGATCGAATTCGAGAC CCTTCTTGG (EcoRI) 1 AGGATGGATCCATAAGTTAC CGATCAG (BamHI) 1 GGCTGGAATTCCTCTGAGGA CGCCCTCAC (EcoRI) 1 GCCGAAGATCTATCGGACAT AGACCTC (Bgl II) 2 CAGACGACGGATCCCCTTGG ATATAGCCTG (BainHI)

(SEQ.

ID.

NO.

71) 72) 73) 74) 76) 77) 78) 79) 81) 82) 83) 84) R 76 B (SEQ. ID. NO. 86) WO 96/10580 PCT/US95/13102 44 D 75 B 5'NC GGCCGAATTCAGGCAGACCA

CATATGTGGTCGATGCCATG

(EcoRI) (SEQ. ID. NO. 87)

D

R

D

R

D

R

D

72 71 63 61 60 59 50 49 48 47 46 1 GCAGGTGTGCCTGGATCCGG CAAGT (BamHI) 1 GTTAGAATTCCGGCCCAGCT GTGGTAGGTC (EcoRI) 1 CCGTCCGATTGGTCTGTATG

CAGG

1 TACCAGTTTACTGCAGGTGT

GC

1 CAAGCCGATGTGGACGTTGT

CG

2,3 GGCGCTGGGCCTGGTCACGC

CAAG

1 GCAGAAACTAGTGTTGACCC

AG

2 TAGGTCTACGACGTGAGGCA

AC

1 TACAATCTTTCAGGAAGAAG

G

1 CCCACACTCCTCCATAATAG

C

1 GATAGTGCTTTGCAGTGAGT

ACCG

The abbreviations to the left

(SEQ.

ID.

NO. 88) NO. 89) NO. NO. 91) NO. 92) NO. 93) NO. 94) NO. NO. 96) NO. 97) NO. 98) of the sequences represent the following: R and D refer to reverse and forward primers, respectively; B and S refer to sequences derived from the Burma-121 Strain of Hepatitis E and the Strain of Hepatitis E, respectively; 5'NC and 3'NC 3 refer to 5 prime and 3 prime non-coding regions of the HEV genome, respectively; and 1, 2 and 3 refer to sequence derived from open reading frames 1, 2 or 3, respectively.

The symbol to the right of some sequences shown indicates insertion of an artificial restriction site into these sequences.

WO 96/10580 PCTIUS95/13102 45 For cloning of PCR fragments, EcoRI, BamHI, or BglII restriction sites preceded by 3-7 nt were added to the 5' end of primers.

RT-PCR. The usual 100-Ai RT-PCR mixture contained template, 10 mM Tris-HCL (ph 50 mM KC1, mM MgCl 2 all four dNTPs (each at 0.2 mM), 50 pmol of direct primer, 50 pmol of reverse primer, 40 units of RNasin (Promega), 16 units of avian myeloblastosis virus reverse transcriptase (Promega), 4 units of AmpliTaq (Cetus), under 100 Al of light mineral oil. The mixture was incubated 1 h at 420C and then amplified by 35 PCR cycles; 1 min at 94 0 C, 1 min at 45 0 C, and 1 min at 72 0

C.

The PCR products were analyzed on 1% agarose gels.

Cloning of PCR Fragments. PCR fragments containing restriction sites at the ends were digested with EcoRI and BamHi or EcoRI and BglII restriction enzymes and cloned in EcoRI/BamHI-digested pBR322 or pGEM- 3Z (Promega). Alternatively, PCR fragments were cloned into pCR1000 (Invitrogen) using the TA cloning kit 2 (Invitrogen).

Sequencing of PCR Fragments and Plasmids.

PCR

fragments were excised from 1% agarose gels and purified by Geneclean (Bio 101, La Jolla, CA). Double-stranded

PCR

fragments were sequenced by using Sequenase (United States S Biochemical) as described in Winship, P.R. (1984), Nucleic Acids Rev., 17:1266. Double-stranded plasmids purified through CsC1 gradients were sequenced with a Sequenase kit (United States Biochemical).

Computer Analysis of Sequences. Nucleotide sequences of HEV strains were compared using the Genetics 3 Computer Group (Madison, WI) software package (Devereaux, J. et al. (1984), Nucleic Acids Rev., 12:387-395, version on a VAX 8650 computer (at the National Cancer Institute, Frederick,

MD)).

WO 96/10580 PCTUS95/13102 46 EXAMPLE 2 Construction of a Recombinant Expression Vector. P63-2.

A plasmid containing the complete ORF-2 of the genome of HEV strain SAR-55, Tsarev, S.A. et al. (1992), Proc. Natl. Acad. Sci. USA, 89:559-563), was used to obtain a restriction fragment NruI-BglII. NruI cut the HEV cDNA five nucleotides upstream of the ATG initiation codon of ORF-2. An artificial Bgl II site previously had been placed at the 3' end of HEV genome just before the 1 poly A sequence (Tsarev, S.A. et al. (1992), Proc. Natl.

Acad. Sci. USA, 89:559-563). To insert this fragment into pBlueBac-Transfer vector (Invitrogen) a synthetic polylinker was introduced into the unique NheI site in the vector. This polylinker contained Bln I and Bgl II sites which are absent in both HEV cDNA and pBlueBac sequences.

The NruI-BglII ORF-2 fragment was inserted in Bln I-BglII pBlueBac using an adapter as shown in Fig. 1.

EXAMPLE 3 Expression of P63-2 in SF9 Insect Cells.

p63-2 and AcMNPV baculovirus DNA (Invitrogen) were cotransfected into SF9 cells (Invitrogen) by the Ca precipitation method according to the Invitrogen protocol By following this protocol; the AcMNPV baculovirus DNA can produce a live intact baculovirus which can package p63-2 to form a recombinant baculovirus. This recombinant baculovirus was plaque-purified 4 times. The resulting recombinant baculovirus 63-2-IV-2 was used to infect SF9 cells.

SDS-PAGE and Western blot. Insect cells were resuspended in loading buffer (50 mM Tris-HC1, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromphenol blue and 10% glycerol) and SDS-polyacrylamide gel electrophoresis was performed as described, Laemmli, U.K. (1970), Nature, 227:680. Gels were stained with coomassie blue or proteins were electroblotted onto BA-85 nitrocellulose filters Schuell). After transfer, nitrocellulose (Schleicher Schuell). After transfer, nitrocellulose WO 96/10580 PCT/US95/13102 -47 0 membranes were blocked in PBS containing 10% fetal calf serum and 0.5% gelatin. As a primary antibody, hyperimmune serum of chimpanzee-1313 diluted 1:1000 was used. As a secondary antibody, phosphatase-labeled affinity-purified goat antibody to human IgG (Kirkegaard Perry Laboratories, Inc.) diluted 1:2000 was used.

Filters were developed in Western blue stabilized substrate for alkaline phosphatase (Promega). All incubations were performed in blocking solution, and washes were with PBS with 0.05% Tween-20 (Sigma).

Expression of HEV ORF-2. The major protein synthesized in SF9 cells infected with recombinant baculovirus 63-2-IV-2 was a protein with an apparent molecular weight of 74 KD (Fig. 2A, lane This size is a little larger than that predicted for the entire ORF-2 (71 KD).

1 The size difference could be due to glycosylation of the protein since there is at least one potential site of glycosylation (Asn-Leu-Ser) in the N-terminal part. This protein was not detected in noninfected cells (Figure 2A, lane 1) or in cells infected with wild-type nonrecombinant 2 baculovirus (Figure 2A, lane In the latter case, the major protein detected was a polyhedron protein. When the same lysates were analyzed by Western blot (Figure 2B) with serum of chimp-1313 (hyperimmunized with HEV), only proteins in the recombinant cell lysate reacted (lane 3) 2 and the major band was again represented by a 74 KD protein (Fig. 2B). Minor bands of about,25, 29, 35, 40-45 and 55-70 kDa present in the Coomassie-stained gel (Fig.

2A, lane 3) also reacted with serum in the Western blot (Figure 2B, lane Some of the bands having molecular weights higher than 74 KD result from different extents of glycosylation while the lower molecular weight bands could reflect processing and/or degradation. Serum drawn from Chimp-1313 prior to inoculation with HEV did not react with any of the proteins by Western blot.

WO 96/10580 PCT/US95/13102 48 0 EXAMPLE 4 Immunoelectron Microscopy of Recombinant Infected SF9 Cells.

5x10 6 recombinant infected SF9 cells were sonicated in CsCl (1.30 g/ml) containing 10 mM Tris-HC1, pH 7.4, 0.3% sarcosyl and centrifuged 68 h, at 40,000 rpm 50 ul of the fraction, which had the highest ELISA response and a buoyant density of 1.30 g/ml was diluted in 1 ml PBS and 5 ul of chimp-1313 hyperimmune serum was added. The hyperimmune serum was prepared by 0 rechallenging a previously infected chimp with a second strain of hepatitis E (Mexican HEV). Samples were incubated 1 h at room temperature and then overnight at 4"C. Immune complexes were precipitated using a rotor at 30,000 rpm, 4'C, 2 h. Pellets were resuspended 1 in distilled water, negatively stained with 3% PTA, placed on carbon grids and examined at a magnification of 40,000 in an electron microscope EM-10, Carl Zeiss, Oberkochen, Germany.

Detection of VLPs. Cell lysates from insect 2 cells infected with wild-type or recombinant baculovirus 63-2-IV-2 were fractionated by CsC1 density centrifugation. When fractions of the CsC1 gradient from the recombinant infected insect cells were incubated with Chimp-1313 hyperimmune serum, two kinds of virus-like 2 particles (VLP) covered with antibody were observed in the fraction with buoyant density of 1.30 g/ml: first (Fig.

antibody covered individual particles that had a size (30 nm) and morphological structure suggestive of HEV, second (Fig. 3B), antibody-coated aggregates of particles smaller than HEV (about 20 nm) but which otherwise resembled HEV. Direct EM showed the presence of a very heterogenous population of objects including some of 30 and 20 nm in diameter respectively, which looked like virus particles but, in the absence of bound antibody, could not be confirmed as HEV. A number of IEM RECTIFIED SHEET (RULE 91)

ISA/EP

WO 96/10580 PCTIUS95/13102 49 0 experiments suggested that at least some of the protein(s) synthesized from the ORF-2 region of the HEV genome, had assembled into a particulate structure. It was observed that insect cells at a later stage of infection, when the proportion of smaller proteins was higher, consistently gave better results in ELISA. Therefore, unfractionated lysates of recombinant insect cells from a later stage of infection were used as antigen in ELISA in subsequent tests.

EXAMPLE Detection by ELISA Based on Antigen from Insect Cells Expressing Complete ORF-2 of Anti-HEV Following Infection with Different Strains of HEV.

5x10 6 SF9 cells infected with 63-2-IV-2 virus were resuspended in 1 ml of 10 mM Tris-HCl, pH 7.5, 0.15M NaCl then were frozen and thawed 3 times. 10 ul of this suspension was dissolved in 10 ml of carbonate buffer (pH 9.6) and used to cover one flexible microtiter assay plate (Falcon). Serum samples were diluted 1:20, 1:400 and 1:8000, or 1:100, 1:1000 and 1:10000. The same blocking and washing solutions as described for the Western blot were used in ELISA. As a secondary antibody, peroxidaseconjugated goat IgG fraction to human IgG or horse radish peroxidase-labelled goat anti-Old or anti-New World monkey immunoglobulin was used. The results were determined by measuring the optical density at 405 nm.

To determine if insect cell-derived antigen representing a Pakistani strain of HEV could detect anti- HEV antibody in cynomolgus monkeys infected with the Mexican strain of HEV, 3 monkeys were examined (Fig. 4).

Two monkeys cyno-80A82 and cyno-9A97, were infected with feces containing the Mexico '86 HEV strain (Ticehurst, J.

et al. (1992), J. Infect. Dis., 165:835-845) and the third monkey cyno-83 was infected with a second passage of the same strain. As a control, serum samples from cyno-374, infected with the Pakistani HEV strain SAR-55, were tested WO 96/10580 PCT/US9/13102 50 0 in the same experiment. All 3 monkeys infected with the Mexican strain seroconverted to anti-HEV. Animals from the first passage seroconverted by week 15 and from the second passage by week 5. Interestingly, the highest anti-HEV titer among the 4 animals, was found in cyno-83, inoculated with the second passage of the Mexican strain.

Cynos inoculated with the firstsecond passage of the Mexican strain.

Cynos inoculated with the first passage of the Mexican strain developed the lowest titers while those inoculated with the first passage of the Pakistani strain developed intermediate titers.

EXAMPLE 6 Specificity of Anti-HEV ELISA Based on Antigen from Insect Cells Expressing Complete ORF-2.

To estimate if the ELISA described here specifically detected anti-HEV to the exclusion of any other type of hepatitis related antibody, serum samples of chimps were analyzed, in sets of four, infected with the other known hepatitis viruses (Garci, P. et al. (1992), J.

Infect. Dis., 165:1006-1011; Farci, P. et al. (1992), Science (in press); Ponzetto, A. et al. (1987) J Infect.

Dis., 155: 72-77; Rizzetto; m.et al. (1981) Hepatology 1: 567-574; reference for chimps 1413, 1373, 1442, 1551 (HAV); and for chimps 982, 1442, 1420, 1410 (HBV); is unpublished data from Purcell et al) (Table Samples of pre-inoculation and 5 week and 15 week post-inoculation sera were analyzed in HEV ELISA at serum dilutions of 1:100, 1:1000 and 1:10000. None of the sera from animals infected with HAV, HBV, HCV and HDV reacted in the ELISA for HEV antibody, but all 4 chimps inoculated with HEV developed the IgM and IgG classes of anti-HEV.

Table 1. Serological assay of anti-HEV antibody in chimpanzees infected with different hepatitis viruses (Hepatitis A, B, C, D, E) chimp inocu- week of preserum weeks post-inoculation lated seroconvirus version 5 15 20/25 for inoculated IgG IgM IgG IgM IgG IgM IgG IgM virus Chimp-1413 HAV 5 Chimp-1373 Chimp-1442 Chimp-1451 Chimp-982 Chimp-1442 Chimp-1420 Chimp-1410 Chimp-51 Chimp-502 Chimp-105 Chimp-793 Chimp-904 Chimp-814 Chimp-800 Chimp-29 Chimp-1310 Chimp-1374 Chimp-1375

HAV

HBV

HCV

HDV

HEV

1:10,000 1:100 1:10,000 1:8000 1:8000 1:8000 1:400 1:400 Table 1 (cont'd.) Chimp-1313 HEVist** 5 Chimp-1313 HEV2nd°** 0.5 1:100 1:10,000 1:100 1:1000 1:10,000 1:10,000 Chimp-1374 was positive for IgM anti-HEV three and four weeks post-inoculation (see Fig.5) Chimp-1313 was inoculated with HEV twice. 1st inoculation with pooled samples of 7 Pakistani patients. 2nd inoculation 45 months later with Mexican strain of HEV.

WO 96/10580 PCT/US95/13102 -53 EXAMPLE 7 Determination of the Host Range of the Strain of HEV in Non-Human Primates.

Different primate species were inoculated intravenously with a standard stool suspension of HEV and serial serum samples were collected to monitor for infection. Serum ALT levels were determined as an indicator of hepatitis while seroconversion was defined as a rise in anti-HEV. The results were compared with those obtained in cynomolgus monkeys and chimpanzees.

Both rhesus monkeys inoculated with HEV (Table 2) demonstrated very prominent peaks of alanine aminotransferase activity as well as a strong anti-HEV response. The peak of alanine aminotransferase activity was observed on day 35 for both animals, and seroconversion occurred on day 21. The maximum titer of anti-HEV was reached on day 29. Both African green monkeys used in this study (Table 2) developed increased alanine aminotransferase activity and anti-HEV. Although African green money 230 died 7 weeks after inoculation, proof of infection was obtained before that time. Peak alanine aminotransferase activity for monkey 74 exceeded the mean value of preinoculation sera by about three times and for monkey 230 by about five times. Peaks of alanine aminotransferase activity and seroconversion appeared simultaneously on days 28 and 21 in monkeys 74 and 230, respectively.

WO 96/10580 WO 96110580PCTIUS95/13102 54 Table 2. Biochemical and serologic profiles of HEV infection in eight primate species.

Alanine aminotransferase (units/L) Anti-HEV IRG Day first detected Maximum Animal mean (SD) Day Value (titer) titer Chimpanzee 1374 51(12) 27 114 27(1:400) 1:8000 1375 41(14) 27 89 27(1:400) 1:8000 Cynomolgus monkey 374* 46(20) 26 608 19(1:400) 1:8000 381* 94(19) 35 180 28(1:20) 1:8000 Rhesus monkey 726 43(6) 35 428 21(1:20) 1:8000 938 29(10) 35 189 21(1:20) 1:8000 African green monkey 74 72(21) 28 141 28(1:400) 1:8000 230 102(45) 21 334 21(1:8000) 1:8000 Pigtail macaque 98 37(8) 21 47 21(1:.400) 1:8000 99 41(6) 28 59 21(1:400) 1:8000 Tamarin 616 28(7) 70 41 636 19(4) 7, 56 Squirrel monkey 868 90(35) 40 355 41(1:20) 1:20 869 127(63) 42 679 35(1:20) 1:20 Owl monkey 924 41(7) 35 97 21(1:20) 1:8000 925 59(6) 49, 91 t 78,199t 21(1:20) 1:8000 NOTE. no anti-HEV detected.

Previously studied using fragments of HEV proteins expressed in bacteria as antigen [18].

t Biomodal elevation of alanine aminotransferase.

SD standard deviation.

WO 96/10580 PCT/US95/13102 55 0 Pigtail macaque 99 demonstrated an increase in alanine aminotransferase activity 3 SD above the mean value of preinoculation sera, while pigtail macaque 98 did not. However, both monkeys seroconverted on day 21 and the anti-HEV titers were equivalent to those of the chimpanzees and Old World monkeys. Because of the low peak alanine aminotransferase values in the pigtail macaques, the possibility of immunization instead of infection with HEV cannot be completely ruled out.

However, immunization is unlikely for several reasons.

First, immunization in either of 2 tamarins, which are only one-fourth as large as the pigtail macaques but received the same amount of inoculum was not observed.

Second, it is well known that the amount of HEV excreted in feces is usually very small, and 0.5 mL of the 1 suspension of feces used in this study is unlikely to contain an amount of antigen sufficient to immunize an animal, especially when inoculated intraveneously.

Neither tamarin inoculated in this study had a significant rise in alanine aminotransferase activity or development of anti-HEV (Table Therefore, these animals did not appear to be infected. The squirrel monkeys did develop anti-HEV, but at significantly lower levels than chimpanzees or Old World monkeys (Table 2).

In addition, seroconversion occured later in other 2 animals. Squirrel monkey 868 seroconverted on day 41 and 869 on day 35. The anti-HEV titer was not 1:20 at any time during 3 months of monitoring and clearly was waning in both animals after reaching a peak value on days 47-54. However, the increases in alanine aminotransferase activity were rather prominent in both animals and were temporally related to the time of seroconversion.

The owl monkeys responded to HEV infection about as well as the Old World monkey species (Table Both owl monkeys seroconverted on day 21 and by day 28 the anti-HEV titer had reached a value of 1:8000. Alanine amino-transferase activity peaked on day 35 in owl monkey WO 96/10580 PCT/US95/13102 56 0 924 but not until day 49 in monkey 925.

EXAMPLE 8 Detection of IgM and IqG Anti-HEV in Chimps.

In both chimps, the serum ALT levels increased about 4 weeks post-inoculation (Table 2, Fig. Both chimps seroconverted at the time of ALT enzyme elevation or earlier (Fig. 5A, 5C). Levels of IgM anti-HEV also were determined for the chimps. In chimp-1374, the titer of IgM anti-HEV (Fig 5B) was not as high as the IgG titer (Fig 5A) and waned over two weeks. Although both IgG and IgM antibodies were first detected for this animal on day the titer of IgM anti-HEV was the highest while the titer of IgG was the lowest on that day, but then rose and stayed approximately at the same level for more than three months. In chimp-1375, only IgM anti-HEV was detected on day 20 (Fig. 5D). The titer was higher than in chimp-1374 and IgM anti-HEV was detected during the entire period of monitoring. IgG anti-HEV was first observed in this animal on day 27 (Fig. 5C) and remained at approximately the same level throughout the experiment.

20 EXAMPLE 9 Comparison of ELISA Based on Complete ORF-2 Protein Expressed in Insect Cells With That Based on Fraqments of Structural Proteins Expressed in E. coli.

To estimate if expression of the complete ORF-2 region of the HEV genome in eukaryotic cells had any advantage over expression of fragments of structural proteins in E. coli, we used the former antigen in ELISA to retest cynomolgus monkey sera that had been analyzed earlier (Tsarev, S.A. et al. (1992), Proc. Natl. Acad. Sci USA, 89:559-563; and Tsarev, S.A. et al. (1993) J. Infect.

Dis. (167:1302-1306)), using the antigen fragments expressed in bacteria (Table 3).

WO 96/10580 PCT/US95/13102 57 0 Table 3. Comparison of ELISA based on antigen from insect cells expressing complete ORF-2 with that based on antigen from E.coli expressing fragments of structural proteins Cyno antigen derived from antigen derived from bacterial cells insect cells (Portion of ORF-2)* (Complete ORF-2) anti-HEV day anti- first HEV first detected max.

detected day titer titer Cyno-376 28 21 1:400 1:8000 Cyno-369 54 40 1:100 1:8000 Cyno-374 19 19 1:400 1:8000 Cyno-375 26 26 1:400 1:8000 Cyno-379 21 19 1:100 1:8000 Cyno-381 28 28 1:400 1:8000 'The sera were also tested with less sensitive ORF-3 antigen Tsarev, S.A. et al. (1993), J. Infect. Dis. (167:1302- 1306) For 3 of the 6 monkeys examined by ELISA, the antigen expressed in insect cells detected seroconversion earlier than the antigen expressed in E. coli. Using the insect cell-derived antigen, we were able to detect anti-HEV antibody in sera from all six monkeys at the highest dilution tested (1:8000). With E. coli-cell derived antigen (Burma Strain) no information about anti-HEV titers were obtained, since all sera were tested only at a dilution of 1:100 (Tsarev, SA et al (1992) Proc. Nat.

Acad. Sci. USA; 89:559-563; Tsarev et al. (1993) J.

Infect. Dis. (167:1302-1306)).

In another study, hepatitis E virus, strain SARwas serially diluted in 10-fold increments and the 10 through 10- 5 dilutions were inoculated into pairs of cynomolgus monkeys to titer the virus. The serum ALT levels were measured to determine hepatitis and serum antibody to HEV was determined by the ELISA method of the present invention (data in figures) or by Genelab's ELISA (three WO 96/10580 PCTIUS95/13102 58 0 ELISAs, each based on one of the antigens designated 4-2, 3-2 and 612 in Yarbrough et al. Virol., (1991) 65:5790-5797) (data shown as positive or negative test at bottom of Figures 6 All samples were tested under code.

The ELISA method of the present invention detected seroconversion to IgG anti-HEV in all cynos inoculated and all dilutions of virus.

In contrast, Genelab's results were strikingly variable, as summarized below.

Table 4.

ELISA of Dilution Present of Virus Genelab's ELISA Invention 10-1 did not test positive 10- 2 positive for both animals, limited duration positive 3 negative for both animals positive 4 Cyno 389: positive for IgM and IgG positive Cyno 383: negative positive 5 Cyno 386: negative positive Cyno 385: positive positive Since Cyno 385 (10 5 was positive in ELISA tests both by Genelabs and the present invention, the 10 4 (ten times more virus inoculated) and 10 3 (100 times more virus inoculated) would also have been expected to be positive.

The present invention scored them as positive in contrast to Genelab's ELISA test which missed both positives at 10 3 and one at 10 4 even though the ALT levels of Cyno 383 and 393 suggested active hepatitis. Therefore, the data support the advantages of the present ELISA method over the prior art methods of detecting antibodies to HEV.

WO 96/10580 PCTIUS95/13102 59 0 EXAMPLE Comparison Of ELISAs Based On Recombinant ORF-2 Antigens Consisting Of Either A 55 kDa Protein Expressed From The Complete ORF-2 Region Of The Pakistani SAR-55 Strain Of HEV Or Of Shorter Regions Of ORF-2 Expressed As Fusion Proteins In Bacteria.

As described in Example 3 and as shown in Figures 2A and 2B, a number of proteins of varying molecular weights are expressed in insect cells infected with the recombinant baculovirus containing the complete ORF-2. A protein with a molecular weight of approximately kDa was partially purified from 5x10 8 SF-9 cells harvested seven days post-inoculation as follows: The infected cells were centrifuged, resuspended in 10 ml of mM Tris-HCl (pH 50 mM NaC1, containing 40 pg/ml of phenylmethylsulfonyl fluoride (Sigma, St. Louis, 1 Missouri), sonicated to disrupt the cells and the lysate was centrifuged at 90,000xg at 4 0 C for 30 min. The supernatant was loaded onto a DEAE-sepharose CL-6B (Pharmacia, Uppsala, Sweden) column equilibrated with mM Tris-HCl (pH 50 mM NaC1. The column was washed 2 with loading buffer and the 55 kDa protein was eluted in mM Tris-HCl (pH 8.0) 250 mM NaC1. Fractions containing the 55 kDa protein were combined and the protein was precipitated by addition of 3 g of (NH 4 2

SO

4 to 10 ml of the protein solution. The protein pellet was dissolved in 2 10 mM Tris-HCl (pH 50 mM NaC1. The 55 kDa protein was then used as the insect cell-expressed HEV antigen in ELISA in comparison testing against ELISAs based on either one of two HEV antigens expressed in bacteria, (3-2 (Mexico) (Goldsmith et al., (1992) Lancet, 339:328-331) or SG3 (Burma) (Yarbough et al., (1993) Assay development of diagnostics tests for hepatitis E. In "International Symposium on Viral Hepatitis and Liver Disease.

Scientific program and abstract volume." Tokyo:VHFL, p 87, Abstract 687). These bacterial antigens were fusion 3 proteins of the 26 kDa glutathione-S-transferase (GST) and either the antigenic sequence 3-2 consisting of 42 WO 96/10580 PCTIUS95/13102 0 amino acids located 6 amino acids upstream of the Cterminus of ORF-2 (Yarbough et al., (1991) J. Virol., 65:5790-5797) or the 327 C-terminal amino acids of ORF-2 (Yarbough et al., (1993)). The ELISAs were carried out as follows.

5 Sixty ng per well of the 55 kDa protein or 200 ng per well of the fusion antigens in carbonate buffer (pH 9.6) were incubated in wells of a polystyrene microtiter assay plate (Dynateck, S. Windham, ME) for 2 h at 37 0

C.

Plates were blocked with PBS containing 10% fetal calf 0 serum and 0.5% gelatin. Serum samples from cynomolgus monkeys inoculated intravenously (note: cynos 387 and 392 were inoculated orally) with a dilution of feces containing the SAR-55 strain of HEV ranging from 10- 1 through 10- 8 as indicated in Table 5 and Figures 7A-7J and 8A-8D were diluted 1:100 in blocking solution.

Peroxidase-conjugated goat anti-human IgM (Zymed, San Francisco, CA) diluted 1:1000 or 1:2000, or peroxidaselabelled goat anti-human immunoglobulin diluted 1:1000 was used as the detector antibody.

20 In all of the ELISA tests except those for the two orally inoculated monkeys, cyno-387 and cyno-392, the kDa and the fusion antigens were tested concurrently in the same laboratory so that the only variable was the antigen used. Criteria for scoring positive reactions in 2 anti-HEV ELISA with the 55 kDa antigen were an optical density value a 0.2 and greater than twice that of a preinoculation serum sample for the same animal. In addition, since both antigens expressed in bacteria were fusion proteins with GST, the optical density of a sample 3 tested with these antigens had to be 3 times higher than that obtained with non-fused GST in order to be considered positive (Goldsmith et al., (1992)).

RESULTS

Both cynomolgus monkeys (377, 378) inoculated with the 10- 1 dilution of the standard HEV fecal suspension had a pronounced increase in ALT activity at 4-5 weeks

I

WO 96/10580 PTU9I3O PCTfUS95113102 61 post-inoculation, indicative of hepatitis (Table Figures 7A and 7B).

Table 5. Summary of biochemical and serological events occurring in cynomolgus monkeys after inoculation with lOr' to 10' dilutions of the standard stock of the SAR-55 HEV inoculum.

'-yno M~lutton ALTi of viral stock pre-inocuinoculum lation. week mean (SD) weeks post-inoculation anti-HEV value weeks post-inoculation anti-HEV was detected with fusion antigen th IgG Ig] peak wa 55 kDa antigen

(UIL)-

lgG Igi s detected wi 4 SG3 3-2(M) SG3 3-2(M) 10-1 10-1 10-2 10-2 10-3 10-5 10-4 10-4 10-1 10-6 10-6 10-7 10-7 10-9 (oral)' (oral)' 76 (39) 50 (9) 62 (14) 121 (21) 89 (20) 29 (8) 60 (7) 41 (4) 59 (32) 31 (4) 60 (4) 36 (3) 102 (16) 57 (4) 33 (3) 56 (4) 32 (4) 49 (6) 4-15t 4-15- 5-15 5-15 5-15* 5-15 6-15 11-15 8-15 4-10 4-5 3-4 3-10 5-13 6-8 8-13 6-15 -7-15 I ALT mean and standard deviation (SD) values of pre-inoculation sera.

t The experiment was terminated after 15 weeks.

A 0LA Table 5 (cont'd.) un *The OD values of pre-inoculation sera of Cyno-3 80, when tested by ELISA with 55 kDa antigen, were twice as high as the mean value of pre-inoculation sera for other cynomolgus monkeys.

'All EUSA tests except for Cyno-387 and Cyno-392 were performed in the same experiments.

-not detected. ND not done.

WO 96/10580 PCT/US95/13102 -64 0 All 3 antigens tested detected IgM anti-HEV in samples taken from cyno-377 3 weeks post-inoculation Table Figure 8A), but IgM anti-HEV was not detected in any samples from the second animal, cyno-378. IgG anti-HEV was detected in both animals with the 55 kDa-based ELISA, but only in cyno-377 with the ELISA based on fusion antigens. Values of OD for IgG anti-HEV were significantly higher than those for IgM anti-HEV. ELISA values obtained with the 55 kDa antigen were also 1 significantly higher than those obtained with either of the two fusion antigens (Figures 7A and 7B). The patterns of the OD values observed in ELISA with antigens from the two sources also differed significantly. In the case of ELISA based on the fusion antigens, positive signals reached a maximum shortly after seroconversion and then 1 waned during the 15 weeks of the experiment. In ELISA based on the 55 kDa antigen, the positive signal reached a maximum shortly after seroconversion and remained at approximately the same high level throughout the experiment (15 weeks).

Elevation in ALT activities in both monkeys (394 and 395) inoculated with a 10 2 dilution of the standard HEV stool suspension was significantly less pronounced at the expected time of hepatitis than in animals inoculated with the ten-fold higher dose Table 5, Figures 7C and 2 7D). Cyno-395 actually had higher ALT activities prior to inoculation as well as at 15 weeks post-inoculation. The latter was probably not related to HEV infection. Weakly positive IgM anti-HEV was detected only in cyno-394 (Figure 8B) and only with ELISA based on the 55 kDa 3 antigen. Both animals were infected, however, since IgG anti-HEV seroconversion was detected in both animals. In cyno-394, anti-HEV IgG was first detected by the 55 kDa antigen at week 3 and one week later with the 3-2(M) antigen. The SG3 antigen did not detect seroconversion in cyno-395 and anti-HEV IgG was detected only with the 55 kDa antigen. Anti-HEV tended to diminish WO 96/10580 PCT/US95/13102 65 0 in titer with time in this animal.

Cyno-380 and cyno-383 were inoculated with a 10- 3 dilution of the standard HEV fecal suspension (Table Figures 7E 7F, 8C). Cyno-380 had fluctuating ALT activities before and after inoculation; therefore, ALT levels could not be used to document hepatitis E in this animal. In Cyno-383, a slight rise of ALT activities was observed (Figure 7F), which was coincident with seroconversion and, therefore, might be due to mild hepatitis E. IgM Anti-HEV was not detected in sera from cyno-380 with any of the three antigens. Cyno-380 seroconverted for IgG anti-HEV when tested by ELISA with SG3 but not with 3-2(M) antigen. This animal had preexisting IgG anti-HEV when tested with the 55 kDa antigen, but there was a large increase in IgG anti-HEV starting at week 5 (Figure 7E). Identification of preexisting antibody was reported earlier in sera from another cynomolgus monkey [Ticehurst et al., (1992) J.

Infect Dis., 165:835-845; Tsarev et al., (1993) J. Infect.

Dis., 168:369-378]. Seroconversion occured at the expected time but the levels of IgG anti-HEV in samples from cyno-383 remained low and detectable only with the kDa antigen.

Cyno-389 and cyno-393 were inoculated with a 10 4 dilution of the standard HEV fecal suspension (Figures 7G, 7H, 8D, Table Neither animal had a significant rise in ALT activities, although the timing of a small but distinct ALT peak in sera of cyno-393 at week 5 (Figure 7H) suggested borderline hepatitis. ELISA based on the SG3 or 3-2(M) antigens scored both animals as negative for HEV infection. In contrast, the 55 kDa antigen detected IgM anti-HEV in sera of cyno-389 at weeks 6-8 post-inoculation (Figure 8D) and IgG anti-HEV from week 6 through week 15 in both animals.

Neither animal inoculated with the 10 5 dilution 3 of the standard fecal suspension developed a noticeable rise in ALT activities (Figure 71, 7J, Table but, in WO 96/10580 PCTIUS95/13102 66 0 cyno-386, IgM and IgG anti-HEV were detected at weeks 8-13 and 8-15 respectively with the 55 kDa antigen (Figure 7J, 8E). Cyno-385 anti-HEV IgG was detected with the 55 kDa and the 3-2(M) antigen but not with SG3 antigen. In contrast to previous patterns, IgG anti-HEV was detected with a fusion antigen four weeks earlier and at higher levels than with the 55 kDa antigen.

None of the animals inoculated with dilutions of the standard HEV fecal suspension in the range of 10--10 8 1 developed antibody to any of the three HEV antigens.

Increased ALT activities were not observed in those animals, except for one rather prominent peak of ALT activity at week 9 in cyno-400 (Table However, the absence of seroconversion in this animal indicated that this peak probably was not related to HEV infection.

With respect to the two cynomolgus monkeys (387 and 392) inoculated orally with the 10-1 dilution of the fecal suspension, neither monkey was infected since ALT levels did not rise and ELISA performed with the 3- 2(M) and 55 kDa antigens did not detect seroconversion to HEV (Table Finally, serological evidence for HEV infection was found in all animals inoculated with decimal dilutions of the fecal suspension through 10-5; none of the animals receiving higher dilutions had such evidence. Prominent 2 hepatitis, as defined by elevated ALT, was observed only in the two monkeys infected with the 10-1 dilution.

Significantly lower elevations of ALT activities were observed in some animals inoculated with higher dilutions of the fecal suspension while, in others, elevations were 3 not found. Considered alone, these low ALT rises were not diagnostic of hepatitis. However, the coincidence of seroconversion and appearance of these ALT peaks suggests the presence of mild hepatitis in these animals. Anti-HEV IgG seroconversion was detected in all animals inoculated 3 with dilutions of fecal suspension ranging from 10-1 -10 5 A tendency toward lower levels of IgG anti-HEV and delayed WO 96/10580 PCT/US95/13102 67 0 seroconversion was observed in animals inoculated with higher dilutions of the stock.

In sum, the 55 kDa Pakistani ORF-2 antigen was more efficient than either the 3-2(M) or SG3 antigen for detecting IgM and IgG anti-HEV in cynomolgus monkeys infected with the Pakistani strain of HEV. For example, for all animal sera except those from cyno-385, detection of IgG or IgM anti-HEV by ELISA was more efficient with the 55 kDa antigen than with either the 3-2(M) or SG3 1 antigen. ELISA with the 55 kDa antigen produced internally consistent and reproducible results, detecting IgG anti-HEV in all ten animals inoculated with a fecal dilution of 10 5 or lower. The magnitude of ELISA signals also decreased as the inoculum was diluted. The fusion antigens did not produce consistent results between the 1 pairs of animals. Only one of each pair of animals inoculated with the 10- 1 10- 2 10-, or 10 5 dilution showed seroconversion to IgG anti-HEV, and only a single seroconversion for IgM anti-HEV was detected with these antigens. Neither of the animals inoculated with the 2 dilution of the original inoculum seroconverted to either of the two fusion antigens even though sera from one animal (cyno-393) had sustained high levels of anti-HEV IgG when assayed with the 55 kDa antigen. Although, as discussed above, ELISA for IgM anti-HEV was significantly 2 less sensitive than ELISA for cynomolgus IgG anti-HEV, the 55 kDa antigen was able to detect anti-HEV IgM in more animals than the 3-2(M) or SG3 antigen. In sum, a definitive conclusion about the infectious titer of the Pakistani viral inoculum used in this study could not be 3 made with the combined data from the 3-2(M) and SG3 (B) based ELISA but could be made with data obtained with the kDa Pakistani ELISA alone.

With respect to cyno-385, the difference in anti-HEV IgG detection between the two test results was four weeks. These data suggest the presence of a distinct epitope in the 3-2(M) antigen recognized by this animal WO 96/10580 PCT/US95/13102 68 0 that is absent in the larger 55 kDa and SG3 antigens.

When total insect cell lysate, which contained both complete ORF-2 (75 kDa) and 55 kDa proteins, was used as antigen to retest these samples, the results were the same as when 55 kDa was used alone. This finding suggests that the 55 kDa protein may not lack 3-2 epitope amino acids but rather that the conformation of the 3-2 epitope sequence differed among all three antigens used in this study. Finally, it is interesting to note that despite the fact that antigen SG3 contained a longer portion of ORF-2 and included the entire sequence of epitope 3-2, it did not detect more positive sera than the 3-2(M) antigen.

EXAMPLE 11 Determination of the Infectious Titer of the HEV SAR-55 Viral Stock BY RT-PCR Knowledge of the infectious titer of inocula is critical for interpretation of much of the data obtained in experimental infections of animal models. However, until now the infectious titer of an HEV viral stock has not been reported. Ten-fold dilutions of the fecal suspension containing the SAR-55 strain of HEV were extracted and RT-PCR amplification was performed as follows to determine the highest dilution in which HEV genomes could be detected. 200 ul of fecal suspension was mixed with 0.4 ml of 1.5M NaC1 plus 15% polyethylene glycol (PEG) 8000 and kept overnite at 4 0 C. Pellets were collected by centrifugation for 3 minutes in a microcentrifuge (Beckman, Palo Alto, CA) at 16,000g and dissolved in 475 ul of solution containing 4.2M guanidine thiocyanate, 0.5% N-lauroylsarcosine, 0.25M TRIS-HC1 (pH 0.15 M dithiothreitol (DTT), and 1.Og of tRNA.

Fifty microliters of 1M TRIS-HC1 (pH 100 mM EDTA, and 10% SDS was then added. The RNA was extracted twice with phenol-chloroform at 65 0 C, followed by chloroform extraction at room temperature. To the upper phase, 250 AL of 7.5 M ammonium acetate was added, and WO 96/10580 PCT/US95/13102 69 0 nucleic acids were precipitated with 0.6mL of 2-propanol, washed with 75% ethanol, washed with 100% ethanol, and used for reverse transcription (RT) PCR.

For detection of the HEV genome, two sets of nested primers were used that represented sequences from the 3' region (ORF-2) of the SAR-55 genome. Primers for reverse transcription and the first PCR are shown as SEQ ID NO:99: GTATAACGGATCCACATCTCCCCTTACCTC and SEQ ID NO:100: TACAGATCTATACAACTTAACAGTCGG respectively. Primers for the second PCR are shown as SEQ ID NO: 101: GCGGCAGATCTCACCGACACCATTAGTAC and SEQ ID NO:102: TAACCTGGATCCTTATGCCGCCCCTCTTAG respectively. The RNA pellet was dissolved in 20 AL of 0.05 M TRIS-HC1 (pH 7.6), 0.06 M KC1, 0.01 M MgCl,, 0.001 M DTT, 40 units of RNasin (Promega Biotec, Madison, WI), 16 units of avian myeloblastosis virus reverse transcriptase (Promega Biotec), and 10 pmol of reverse primer and incubated 1 hour at 42 0 C. To 20 AL of reverse transcriptase mixture was added 100 pL of 0.01 M TRIS-HC1 (pH 0.05 M KC1, 0.0025 M MgCI 2 0.0002 M each dNTP, 50 pmol of direct primer, 50 pmol of reverse primer, and 4 units of AmpliTaq (Perkin-Elmer Cetus, Norwalk, CT) under 100 AL of light mineral oil. The HEV cDNA was amplified by 35 cycles of PCR:1 min at 94 0 C, 1 min at 55 0 C, 1 min at 72 0 C. The products of PCR were analyzed on 1% agarose gels. Then /L of this mixture was used for the second round of amplification under the same conditions, except the extension time was increased to 3 min.

The RT-PCR products produced in all dilutions of the standard HEV feces in the range from 101 to 10 (Figure 9) were separated on a 2% agarose gel and were detected by ethiduim bromide staining of the gel. A decrease in the amount of the specific PCR product at higher dilutions was observed and the highest dilution of the 10% fecal suspension in which the HEV genome was 3 detected was 10 5 Therefore, taking into account the dilution factor, the HEV genome titer was approximately WO 96/10580 PCT/US95/13102 70 67 per gram of feces.

In addition, only those dilutions that were shown by RT-PCR to contain the HEV genome were infectious for cynomolgus monkeys. Therefore, the infectivity titer of the standard fecal suspension and its genome titer as detected by RT-PCR were approximately the same. A similar correlation between RT-PCR and infectivity titer was found for one strain of hepatitis C virus [Cristiano et al., (1991) Hepatology, 14:51-55; Farci et al., (1991) N. Engl.

1 J. Med., 25:98-104; Bukh et al., (1992); Proc. Natl. Acad.

Sci 89:187-191) EXAMPLE 12 Active Immunization Using The ORF-2 Protein As A Vaccine And Passive Immunization With Anti-HEV Positive Convalescent Plasma Cynomolgus monkeys (Macaca fascicularis) that were HEV antibody negative in an ELISA based on the 55 kDa ORF-2 protein were individually housed under BL-2 biohazard containment and a suspension (in fetal bovine serum) of feces containing the Pakistani HEV strain SAR-55, diluted to contain 10,000 or 1,000 CID 50 was used for intravenous inoculation of animals.

For active immunization studies, baculovirus recombinant-expressed 55 kDa ORF-2 protein was purified from 5x10 8 SF-9 cells harvested 7 days post-inoculation as described in Example 10. Three mg of the purified 55 kDa protein were precipitated with alum and eight cynomolgus monkeys were immunized by intramuscular injection with ml of vaccine containing 50 pg of the alum-precipitated kDa protein. Four monkeys received a single dose and four monkeys received two doses separated by four weeks.

Primates were challenged intravenously with 1,000 10,000

CID

50 of HEV four weeks after the last immunization.

Four cynomolgus monkeys served as controls in the active immunization studies. Cyno-412 and 413 received one dose of placebo (0.5 ml of phosphate buffered saline) and cyno-397 and 849 received two doses of WO 96/10580 PCT/US95/13102 71 placebo. The control animals were challenged with 1,000 10,000 CID 0 of HEV.

For passive immunity studies, cyno-384 was infected with 0.5 ml of a 10% pooled stool suspension containing two Chinese HEV isolates, KS1-1987 and KS2-1987 and plasma was repeatedly collected from the animal during convalesence. (Yin et al. (1993) J. Med. Virol., 41:230- 241;). Approximately 1% of the blood of cyno-396 and cyno-399 and 10% of the blood of cyno-401 and cyno-402 was replaced with convalescent plasma from cyno-384 having an HEV antibody titer of 1:10,000. Animals were challenged with 1000 CID 50 of HEV two days after infusion of the plasma. As a control, 10% of the blood of cyno-405 was replaced with anti-HEV negative plasma obtained from cyno- 384 prior to infection with HEV. Cyno-405 was then challenged with 1000 CID, 5 of HEV.

For both the passive and active immunization studies, percutaneous needle biopsies of the liver and samples of serum and feces were collected prior to inoculation and weekly for 15 weeks after inoculation.

Sera were assayed for levels of alanine amino transferase (ALT) with commercially available tests (Metpath Inc., Rockville, MD) and biochemical evidence of hepatitis was defined as a two-fold or greater increase in ALT. Liver biopsies were examined under code and the anti-HEV ELISA utilized was described in Example 10. RNA extraction and RT-PCR were performed as in Example 11 except that RNA from 100 pl of serum or from 100 pl of 10% fecal suspension was extracted with TRIzol Reagent (Gibco BRL, Gaithersburg, Maryland) according to the manufacturer's protocol. For quantification, PCR positive serial sera or feces from each animal were combined and serially diluted in ten-fold increments in calf serum. One hundred gl of each dilution were used for RNA extraction and RT-PCR as described earlier in this Example. The PCR protocol used in this study could detect as few as 10 CID 50 of HEV per ml of serum and as few as 100 CID 50 per gram of feces.

WO 96/10580 PCTIUS9/13102 72 Peak ALT values of weekly serum samples for weeks prior to inoculation and for 15 weeks postinoculation were expressed as ratios (post/pre) for each animal. The geometric mean of the ratios from the control group of animals was compared with that from the passively or actively immunized animals using the Simes test (Simes, R.J. (1986) Biometrika, 73:751-754).

The durations of viremia and virus shedding in feces and the HEV genome titers in the control group of animals were compared with those in passively or actively immunized animals using the Wilcoxon test [Noether, G.

(1967) in Elements of nonparametric statistics (John Wiley Sons Inc., New York), pp. 31-36.]. The same test was used to compare the above parameters between passively and actively immunized animals.

For statistical analysis, serum samples that had HEV genomes in 1 ml of serum were assigned a titer of 1:1 and fecal samples that had <100 HEV genomes in 1 g of feces were assigned a titer of 1:10.

RESULTS

Course of hepatitis E infection in nonimmunized animals.

In 3 of 5 nonimmunized animals that were challenged with HEV, biochemical evidence of hepatitis was documented by at least a two-fold increase in serum ALT values. In 2 two animals, significant increases in ALT activity were not found. However, histopathological data documented hepatitis in all 5 animals as shown in Table 6.

Table 6. Histopathological, biochemical, serological, and virological profiles of vaccinated and control animals challenged with

HEV.

Animal Anti-HEV Cumulative Peak ALT value in U/L HEV HEV genome and positive score of (week) antibody category plasma histopa- titer at the or thology pre- post- time of serum feces kDA (number of inoculation inoculation challenge protein weeks de- week de- mean logo week de- mean loglo (Lg) tected)*. tected titer per tected titer per (duration) ml (duration) gram control 405 412 413 849 397 0 10+ (8) 0 2+ (1) 0 4+ (4) 0 1+ (1) 0 3+ (3) 1% 1+ 67 (0) 34 (0) 44 (0) 79 52 33 (0) 69 (0) 143 (9) 45 (3) 261 (6) 133 (2) 139 (7) 53 (6) 63 (11) <1:10 1-11 (11) <1:10 1-4(4) <1:10 2-7(6) <1:10 1-4(4) <1:10 2-6(5) 1:40 3-5 (3) 1:40 2-4(3) 3 1-11 (11) 5.7 2-5 (4) 1-7 (7) 1-4 (4) 1-7(7) passive IPt 396 399 1-6 (6) 1-4 (4) 0 (0)

M

O I 401 402 10% 10% 0 (0) 0 (0) 55 (0) 59 (0) 45 (5) 35 (2) 1:200 1:200 3 (1) 4-6 (3) 1-3 (3) 2-6 active IPt 003 009 013' 414 398 407 50 50 50 50 2x50 2x50 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0(0) 34 34 44 65 (0) 31 (0) 150 (0) 50 (6) 38 (6) 36 (7) 73 (8) 41 (2) 213 (4) 1:10,000 1:1,000 1:100 1:1,000 1:10,000 1:10,000 2-4 (3) 0 1-2 (2) 2 (1) 0 0 *Necro-inflammatory changes in the live tImmunoprophylaxis *Necro-inflammatory changes rated 1+ hepatitis only during one week.

'Cyno 013 died 9 weeks after challenge.

1:10,0000 r were rated as 4+ and the weekly scores were summed.

were detected during two weeks in cyno-396, however, they were consistent with viral WO 96/10580 PCT/US95/13102 75 0 Necro-inflammatory changes ranged between 1+ and 2+ on a scale of 1+ to 4+ and were temporally associated with elevations of ALT activities in those animals with such elevations.

All control animals seroconverted to HEV weeks post-challenge and developed maximum HEV antibody titers ranging from 1:1,000 to 1:32,000. There was a good correlation between the severity of infection, hepatitis, and the level of anti-HEV response. Cyno-405, which had the highest cumulative score for hepatitis, also had the longest period of viremia and viral excretion and the highest level of anti-HEV (Table The duration of viral shedding in feces was the same as, or longer than, that of the viremia. For all of the control animals, titers of the HEV genome in serum were lower (10 3 10 7 than the titers in feces (10"7-10 7 In all five of these animals, viremia and virus shedding in feces were detected for 4-11 weeks and for an average of 4.2 weeks after seroconversion (range 2-9 weeks).

Passive immunization. Cyno-396 and 399, which had approximately 1% of their blood replaced with anti-HEV positive convalescent plasma, had an HEV antibody titer of 1:40 when it was determined two days post-transfusion (at the time of challenge) (Table A two-fold fall in HEV antibody titer was observed in both animals 1 week post- 2 transfusion and HEV antibodies fell below the detectable level by 2 weeks post-transfusion. Anti-HEV was again detected 5 weeks post-challenge in cyno-396 and 4 weeks post-challenge in cyno-399, indicating that infection with HEV had occurred. The maximum HEV antibody 3 titer (1:8,000) was reached 9-10 weeks post-challenge.

Neither cynomolgus monkey demonstrated a significant elevation of ALT activity after challenge. However, histologic evidence of hepatitis was detected in cyno-396 and the HEV genome was detected in serum and feces from 3 both animals (Table 6).

Cyno-401 and 402 had approximately 10% of their WO 96/10580 PCTAUS95/13102 76 blood replaced with convalescent plasma. Two days posttransfusion, at the time of challenge, the HEV antibody titer in both cynomolgus monkeys was 1:200 (Table 7).

W

LI L0 Table 7. 11EV antibody profiles in control and immunized cynomolans monkeys.

Control 11EV antibody Passively 11EV antibody Actively HEV antobody animals ___immu- titer (week max. nzd titer at max. ie max. max. max.

first de- titer animals the time titer animals titer titer titer tected) (week) of chal- (week (week (week (week lenge after after I1st after 2nd after chal- immuni- immuni- challenge) zation) zation) lenge) cyno-405 1:80 1:32,000 cyno-396 1:40 1:8,000 cyno-003 1:10,000 1:10o,000 (10) cyno-412 1:100 1:10,000 cyno-399 1:40 1:8,000 cyno-009 1:10,000 1:10,000 (1) cyno-413 1:100 1: 10, 000 cyno-401 1:200 1:4,000 cyno-013 1:100 1:10,000 (3) cyno-849 1:100 1:1,000 cyno-402 1:200 1:80 cyno-414 1:1,000 1:1,000 (12) (0) cyno-397 1:100 1:10,000 cyno-398 1:1,000 1:10,000 1:10,000 (0) cyno-407 1:1,000 1:10,000 1:10,000 (0) Anti-HEV was detected continuously in both animals during the 15 weeks after challenge and reached a maximum titer of 1:4,000 in cyno-401 but only 1:80 in cyno-402.

WO 96/10580 PCT/US95/13102 78 of 1:4,000 in cyno-401 but only 1:80 in cyno-402.

Biochemical and histologic analyses did not reveal hepatitis in either animal. However, in both animals, HEV viremia and fecal shedding of virus were observed indicating that infection had occurred (Table Thus, passive immunoprophylaxis that achieved a higher titer of antibody protected cynomolgus monkeys against hepatitis after challenge with HEV.

Active immunization. Four primates immunized with one 50 Ag dose of the 55 kDa protein developed antibody to the recombinant protein ranging in titer from 1:100 to 1:10,000 (Table One (cyno 013) died of an anesthesia accident 9 weeks after challenge and is included in the analyses (Table The four animals that received two doses of the antigen developed HEV antibodies with titers of 1:10,000. Two of the four monkeys died following intravenous challenge with HEV. This may have also been the result of an anesthesia accident but the exact etiology could not be determined. These two monkeys were deleted from further analyses. None of the 6 remaining animals developed abnormal ALT levels or histologic evidence of hepatitis following challenge (Table Cynomolgus monkeys immunized with either 1 or 2 doses of the 55 kDa protein did not develop viremia.

However, 3 of 4 animals that received one dose of the immunogen excreted virus in their feces. In contrast, virus shedding was not observed in either of the two challenged animals that had received two doses of the vaccine.

Most of the actively immunized animals developed higher HEV antibody titers than did passively immunized animals. However, cyno-013 had an HEV antibody titer of 1:100 at the time of challenge, compared with a titer of 1:200 in two animals immunized passively with anti-HEV plasma. Cyno-013, however, demonstrated greater protection against HEV infection than the passively immunized animals. Cyno-009, which had an HEV antibody titer of 1:1,000 at the time of challenge, was completely protected against hepatitis and HEV infection (Table 6).

In contrast, cyno-003 was infected and shed HEV in feces, even though it had an HEV antibody titer of 1:10,000 at the time of challenge. However, neither hepatitis nor viremia was detected in this animal or in other cynomolgus monkeys that received one dose of immunogen and had HEV antibody titers of 1:10,000 or greater.

n Comparison of course of HEV infection in control and immunized animals.

10 As measured by histopathology, all immunized animals, with the exception of one of the passively immunized monkeys, were protected against hepatitis after intravenous challenge with HEV. Comparison of mean values for severity of hepatitis and level of viral replication between the control group and the passively and actively immunized animals indicated that, in general, the severity of infection was inversely related to the HEV antibody titer at the time of challenge and diminished in the order: unimmunized>passive immunization (1%)>passive Simmunization (10%)>active immunization (1 dose)>active 0*0. immunization (2 doses) (Tables However, the number of animals in each of the two subgroups of passively and actively immunized animals was not sufficient to permit 00 statistical analysis. Therefore, statistical analysis was 25 performed for combined passively immunized and combined actively immunized groups respectively in comparison with the combined control groups. The results of this analysis are presented in Table 8, r 4 a&0 Ut 0 0 00 00 000 0 00 0 t3' 00 so* 00 000 o 00 .0 *.so 0 0 0 Table 8 Summuty of mean values of HHV infection in control and immunied animal..

Catemoy Hi.*"fptblogy GM of peak ALT REV HEV genome (numnber) Mean of Weeks tIJL antiboySeuFcs of animals cumulative tr-n-Ps-W iter It the nm ma o, e ne o, Wore culation irio-tieo SCOc tie o number titer number titer tion chalenge of weeks of'weeks Cmoto(S) 3.4 53 125 2.4 <1:10 6 3.3 6.6 6.7 Passive t 0.5+ 0.5 4 9 58 1. 1 40 3 3.5 5 4.9 Passive 10%(2P 0 0 57 40 0.7 1:200 2 2.31 4 5.7 Active I dose (4)t 0 OT 43 47 1.1 1:3,025 0 ]L 1.5 2 Active 2doses 0 0 68 93 1.4 1:10,000 0 <I 0 <2 'beomnetric mean 1 'Pasave andcive immunprohylaxie .P<0.05 y not significaint WO 96/10580 PCItS95/13102 81 0 and they show that the histopathology scores and duration of histologic changes in the control group were statistically different from those of passively or actively immunized animals. The higher post-/preinoculation ratios of peak ALT values in the control group were statistically significant when compared with those of the passively or actively immunized animals, indicating protection against biochemical manifestations of hepatitis in both groups of immunized animals. The duration of viremia and the titer of HEV in the feces were significantly lower in both groups of immunized animals than in the control group. Differences in the duration of virus shedding and titer of HEV in the serum, however, were not statistically different between the control group and the passively immunized group, although these parameters were significantly different when the control group was compared with the actively immunized group.

Significant differences were also found between passively and actively immunized groups of animals for duration of viremia and fecal shedding as well as for HEV titers.

In sum, the results presented in Tables 6-8 show that both passively and actively acquired HEV antibodies protected cynomolgus monkeys against hepatitis following challenge with virulent HEV. Although all 5 nonimmunized cynomolgus monkeys developed histologic evidence of hepatitis when challenged with 1,000 10,000 CID 0 of SARboth animals with passively acquired antibody titers of 1:200 were protected from hepatitis and one of two animals with an antibody titer as low as 1:40 also did not develop hepatitis.

However, it should be noted that actively immunized animals demonstrated complete protection against hepatitis and more effective resistance to HEV infection than did passively immunized animals. For example, in contrast to results obtained from the passively immunized animals, viremia was not detected in actively immunized animals after challenge with HEV. An HEV antibody titer as WO 96/10580 PCTIUS95/13102 82 0 high as 1:10,000 could be achieved in cynomolgus monkeys after one or two immunizations with the recombinant 55 kDa protein. Although one monkey (013) developed a titer of 1:100 after active immunization, this level still prevented hepatitis and viremia.

The active immunization studies also demonstrated that while a single dose of vaccine prevented HEV viremia, viral shedding in feces was still detected.

However, two doses of vaccine were observed to prevent all signs of hepatitis and HEV infection. These results thus suggest that a single dose of vaccine administered, for example, to individuals before foreign travel would protect them from hepatitis E in high risk environments.

Finally, it is noted that the results presented 1 are very similar to results reported previously for passive and active immunoprophylaxis of nonhuman primates against hepatitis A: passive immunoprophylaxis prevented hepatitis but not infection whereas vaccination prevented not only hepatitis but infection with HAV as well (Purcell, R.H. et al. (1992) Vaccine, 10:5148-5149). It is of interest that the study of immunoprophylaxis for HEV presented herein parallels the previous study of immunoprophylaxis against HAV, both in determination of the titer of antibody that protected (<1:100) and in outcome following intravenous challenge with virulent virus. Since other studies have demonstrated efficacy of comparable titers of passively and actively acquired anti- HAV in humans and have confirmed the predictive value of studies of primates in hepatitis research (Stapleton,

J.,

et al. (1985) Gastroenterology 89:637-642; Innis, B.L., et al. (1992) Vaccine, 10: S159), it is therefore highly likely that these results in cynomolgus monkeys will be predictive of protection in humans.

EXAMPLE 13 Direct Expression In Yeast Of Complete ORF-2 Protein And Lower Molecular Weiht Fragments Four cDNA ORF-2 fragments coding for: WO 96/10580 PCTIUS95/13102 83 0 1. complete ORF-2 protein (aa 1-660, MW 70979), fragment 1778-1703. (where the fragment numbers refer to the primer numbers given below) 2. ORF-2 protein starting from 34th aa (aa 34- 660, MW 67206), fragment 1779-1703.

3. ORF-2 protein starting from 96th aa (aa 96- 660, MW 60782), fragment 1780-1703.

4. ORF-2 protein starting from 124th aa (aa 124-660, MW 58050), fragment 1781-1703.

were obtained using PCR by using plasmid P63-2 as template and the synthetic oligonucleotides shown below: SEQ ID NO.:103 (reverse primer #1703) GCACAACCTAGGTTACTATAACTCCCGAGTTTTACC, SEQ ID NO.:104 (direct primer #1778) GGGTTCCCTAGGATGCGCCCTCGGCCTATTTTG, SEQ ID NO.:105 (direct primer #1779) CGTGGGCCTAGGAGCGGCGGTTCCGGCGGTGGT, SEQ ID NO.:106 (direct primer #1780) GCTTGGCCTAGGCAGGCCCAGCGCCCCGCCGCT and SEQ ID NO.:107 (direct primer #1781)

CCGCCACCTAGGGATGTTGACTCCCGCGGCGCC.

All sequences shown in SEQ ID NOs: 103-107 contain artificial sequence CCTAGG at their 5' ends preceded by 4 nucleotides. The artificial sequence was a recognition site for Avr II (Bln I) restriction enzyme.

Synthesized PCR fragments were cleaved with BlnI and cloned in the AvrII site of pPIC9 vector (Figure (Invitrogen). Correct orientation of the fragments was confirmed by restriction analysis, using asymmetric EcoRI site present in ORF-2 sequences and in the vector.

Purified recombinant plasmids pPIC9-1778 (containing 3 fragment 1778-1703); pPIC9-1779 (containing fragment 1779- 1703); pPIC9-1780 (containing fragment 1780-1703) and pPIC9-1781 (containing fragment 1781-1730) were used for transformation of yeast spheroplast (Picha strain) according to Invitrogen protocol. Screening of recombinant clones and analysis of expression were performed using the same protocol. These expressed WO 96/10580 PCT/US95/13102 84 proteins may be used as immunogens in vaccines and as antigens in immunoassays as described in the present application. Finally, those of skill in the art would recognize that the vector and strain of yeast used in the above example could be replaced by other vectors (e.s.

pHIL-Fl; Invitrogen) or strains of yeast (e.g.

Saccharomyces Cerevisiae).

EXAMPLE 14 Purification and Amino Terminal Sequence Analysis of HEV ORF-2 Gene Products Synthesized in SF-9 Insect Cells Infected With Recombinant Baculovirus 63-2-IV-2 As described in Example 10, SF-9 cells were infected with recombinant baculovirus 63-2-IV-2 and harvested seven days post-inoculation. The predominant protein band present on SDS-PAGE of the insect cell lysate was approximately 55 kDa in molecular weight. Further purification of this 55 kDa band was accomplished by ionexchange column chromatography using DEAE-sepharose with a 150-450 mM NaC1 gradient. DEAE fractions were assayed for the presence of the 55 kDa band by SDS-PAGE followed by 2 Coomassie blue staining. The peak fraction was then resolved by polyacrylamide gel electrophoresis in the absence of SDS into three bands of 55 kDa, 61 kDa and a band of intermediate molecular weight. Analysis of each protein band from the polyacrylamide gel by amino-terminal microprotein sequencing revealed that the 55 and 61 kDa proteins shared a unique N-terminus at Ala-112 of SEQ ID NO:2. It is believed that the size differences in the two ORF-2 cleavage products may reflect either different glycosylation patterns or a COOH-terminal cleavage of the larger product.

The third intermediate protein on the polyacrylamide gel was shown to be a baculovirus chitinase protein. The 55 and 61 kDa ORF-2 proteins were resolved into a single symmetrical peak fraction devoid of any chitinase by subjecting peak DEAE fractions to reverse phase HPLC using a micropore system with NaCl and WO 96/10580 PCT/US95/13102 85 0 acetonitrile solvents.

EXAMPLE Direct Expression of 55 and 61 kDa Cleavage Products A cDNA ORF-2 fragment coding for ORF-2 protein starting from the 112th amino acid (amino acids 112-660 of ORF-2) was obtained by PCR using plasmid p63-2 as the template. The cDNA fragment was then inserted into a PBlueBac-3Transfer vector at the BamHI-PstI site in the vector. SF9 insect cells are infected with the recombinant baculovirus generated from this vector and insect cell lysates are analyzed for the presence of the 55 and 61 kDa ORF-2 proteins by Coomassie blue staining of polyacrylamide gels. The directly expressed protein(s) may be used as immunogens in vaccines and as antigens in immunoassays as described herein.

Claims

1. A nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of a hepatitis E virus open reading frame 2 protein.

2. A nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of SEQ ID No. 2.

3. A recombinant vector comprising the nucleic acid molecule according to claim 1 or 2. o,

4. The vector according to claim 3, wherein said vector is a 10 baculovirus vector.

A host cell transformed, transfected or infected in vitro with the vector according to claim 3 or 4. i

6. A method for producing a hepatitis E virus open reading frame 2 protein comprising: culturing a host cell containing a nucleic acid molecule Sconsisting of nucleotides which encode amino acids 112-660 of a hepatitis E virus open reading frame 2 protein under conditions appropriate to cause S* expression of said protein; and obtaining said expressed protein from the host cell.

7. A method for producing a hepatitis E virus open reading frame 2 protein comprising: culturing a host cell containing a nucleic acid molecule consisting of nucleotides which encode amino acids 112-660 of SEQ ID No:2 under conditions appropriate to cause expression of said protein; and obtaining said expressed protein from the host cell.

8. A purified and isolated hepatitis E virus open reading frame 2 protein produced by the method according to claim 6, wherein said protein has VAT:Winword\Violet\PhilRNodelete\38309-95.doc 491210 1 C, NfT-. 3~ 87 a molecular weight of approximately 55 kilodaltons and its amino terminus at amino acid 112 of a hepatitis E virus open reading frame 2 protein.

9. A purified and isolated hepatitis E virus open reading frame 2 protein produced by the method of claim 7, wherein said protein has a molecular weight of approximately 55 kilodaltons and its amino terminus at amino acid 112 of SEQ ID No: 2.

10 *o~ S 4 04 a a A purified and isolated hepatitis E virus open reading frame 2 protein having a molecular weight of approximately 55 kilodaltons and its amino terminus at amino acid 112 of a hepatitis E virus open reading frame 2 protein.

11. A purified and isolated hepatitis E virus open reading frame 2 protein having a molecular weight of approximately 55 kilodaltons and its amino terminus at amino acid 112 of SEQ ID No: 2.

12. A method of detecting antibodies to hepatitis E virus in a biological sample, said method comprising: contacting said sample with the hepatitis E virus protein according to any one of claims 8 to 11 to form an immune complex with the antibodies; and detecting the presence of the immune complex.

13. A kit comprising the protein according to any one of claims 8 to tee. Sb 0 SO C a 000 00 Sa 11.

14. A pharmaceutical composition comprising the protein according to any one of claims 8 to 11 in a suitable excipient, diluent or carrier.

A vaccine for immunizing a mammal against hepatitis E, said vaccine comprising the protein according to any one of claims 8 to 11.

16. Use of a protein according to any one of claims 8 to 11 for the manufacture of a medicament for use in a method of preventing hepatitis E in a mammal, comprising administering the medicament to the mammal in an amount effective to stimulate the production of protective antibodies. S VAT:WinwordViolet\Phil\Nodeletel38309-95.doc 491210 1 88

17. Antibodies having specific binding affinity for a hepatitis E virus open reading frame 2 protein having a molecular weight of approximately kilodaltons and its amino terminus at amino acid 112 of a hepatitis E virus open reading frame 2 protein.

18. The antibodies of claim 17, wherein said antibodies are polyclonal antibodies.

19. The antibodies of claim 17, wherein said antibodies are monoclonal antibodies.

Use of the antibodies according to any one of claims 17 to 19 for 10 the manufacture of a medicament for use in a method of preventing hepatitis E in a mammal, comprising administering the medicament to the mammal in a prophylactically effective amount.

21. A method of detecting hepatitis E virus in a biological sample, said j method comprising: contacting said sample with the antibodies according to any one of claims 17 to 19 to form an immune complex with the hepatitis E virus; and detecting the presence of the immune complex.

22. A purified protein according to claim 8 substantially as hereinbefore described with reference to Example 10 or 14. DATED: 17 September, 1999 PHILLIPS ORMONDE FITZPATRICK Attorneys for: THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES VAT:WinwordViolet\PhilNodelete\38309-95.doc 491210 1